Compositions and methods useful for promoting milk production

Abstract

Methods, agents, and compositions for promoting lactation in a mammal are provided. An agent useful for promoting lactation can comprise an agent that inhibits NOTCH4 activity. The agent can inhibit NOTCH4 activity by binding to ROBO2 and / or by binding to NOTCH4. The agent can inhibit NOTCH4 by competing with ROBO1 for binding to ROBO2, thereby making ROBO1 available to inhibit NOTCH4 activity. The agent can be a soluble ROBO1 extracellular domain or an anti-NOTCH4 antibody that inhibits NOTCH4 activity. The agent can be an RNAi construct that inhibits expression of NOTCH4 or an RNAi construct that inhibits expression of ROBO2. Also provided herein are transgenic mammals that are genetically modified to express a soluble ROBO1 extracellular domain; to inhibit expression of ROBO2; and / or to inhibit expression of NOTCH4. Methods of promoting lactation in such transgenic mammals by administering one or more of the agents disclosed herein are also provided.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 837,590, filed April 23, 2019, which is incorporated herein by reference in its entirety.

[0003] By referencing and incorporating the sequence list provided as a text file

[0004] The sequence list is provided in this document as a text file UCSC-383PRV2 seq list_ST25.txt, created on February 15, 2019, and measuring 130KB in size. The text file is cited in its entirety and as per this document. Background Technology

[0005] The mammary glands or breasts of mammals are dynamic epithelial organs responsible for milk production. 1 The mammary gland begins as a primordium located at the nipple and develops after birth in response to hormonal signals generated during puberty, forming ductal structures that branch into the underlying stromal fat pad. Each duct is double-layered, consisting of an outer layer of basal / myoepithelial cells (referred to as BC in this paper) and an inner layer of luminal cells (referred to as LC in this paper). Lumenal cells can be further subdivided into two subgroups: the ductal subgroup surrounding the lumen and the alveolar subgroup that produces milk-producing alveoli during pregnancy. Figure 1 A). Once offspring are weaned from breast milk, the mammary glands remodel back to their pre-pregnancy state through a process known as regression. Acinar progenitor cells (AVPs) are found within the acinar cell subset. It is currently believed that the development of mammary alveoli during pregnancy is caused by the differentiation of ACPs into milk-producing acinar cells (AVs).

[0006] Notch is a major signaling pathway regulating stem / progenitor cell maintenance and fate determination. There are four types of NOTCH receptors: NOTCH1, NOTCH2, NOTCH3, and NOTCH4—all of which are expressed in the mammary gland. 2 During mammary gland development, Notch signaling promotes luminal cell fate at the expense of basal cell fate. 3-6 Furthermore, inhibition of NOTCH4 activity appears to be essential for mammary alveolar expansion and differentiation, as studies have shown that overexpression of the constitutively active NOTCH4 intracellular domain (ICD) significantly reduces mammary alveolar development. 7-9 This suggests that signaling via NOTCH4 must be suppressed in mammary alveolar progenitor cells in order to differentiate them into mammary alveolar cells.

[0007] The ROBO receptor is a member of a conserved immunoglobulin (Ig) superfamily involved in many developmental processes. It binds to a conserved, secreted glycoprotein extracellular matrix ligand family called SLITs (e.g., SLIT2 and SLIT3 in the mammary gland), which are expressed throughout the mammary epithelium. Figure 1 B) 10,11 This signal transduction system has been shown to regulate cell fate determination in the nervous system of mice and the gut of fruit flies. 12,13 . Summary of the Invention

[0008] To supply breast milk during each pregnancy, significantly accelerated cell growth and differentiation are required. This paper discloses a method to promote accelerated cell growth and differentiation by treating the breast with an agent that affects the signal transduction circuit, thereby inhibiting ROBO2 and subsequently NOTCH4 activation. ROBO1 is expressed in both BC and LC of the primitive mammary gland, but is upregulated in LC during pregnancy. ROBO2 expression is limited to a subset of luminal cells. This paper discloses for the first time the following findings: loss (or deletion) of the Robo1 gene leads to inhibition of mammary alveolar differentiation. This has been confirmed in both the HC11 cell lactation model and in vivo mammary glands. Loss (or deletion) of Robo2 produces opposite phenotypes in both models—namely, larger mammary alveolar differentiation. ROBO1 has been shown to specifically bind to NOTCH4 and inhibit its signaling. ROBO2 has been shown to specifically bind to ROBO1 and prevent ROBO1 from inhibiting NOTCH4. The interaction between ROBO1 and ROBO2 is enhanced by SLIT2. This paper discloses a ROBO1 receptor fragment, which includes a portion of the ROBO1 extracellular domain that inhibits NOTCH4 signaling. The experiments disclosed in this paper demonstrate that SLIT / ROBO signaling regulates mammary alveolar production by controlling NOTCH4 activation and the number of alveolar progenitor cells differentiating into milk-producing alveolar cells.

[0009] Methods, agents, and compositions for promoting milk production in mammals are provided. Agents that can be used to promote milk production may contain agents that inhibit NOTCH4 activity. The agent may be a soluble ROBO1 extracellular domain, or the agent may inhibit NOTCH4 activity by binding to ROBO2 and / or by binding to NOTCH4. The agent may inhibit NOTCH4 by competing with ROBO1 for non-binding to ROBO2, thereby making ROBO1 available for inhibiting NOTCH4 activity. The agent may be an anti-NOTCH4 antibody that inhibits NOTCH4 activity. The agent may be an RNAi construct that inhibits NOTCH4 expression. The agent may be an RNAi construct that inhibits ROBO2 expression. This document also provides transgenic mammals that are genetically modified to express a soluble ROBO1 extracellular domain; inhibit ROBO2 expression; and / or inhibit NOTCH4 expression. Methods for promoting milk production in such transgenic mammals by administering one or more of the agents disclosed herein are also provided. Attached Figure Description

[0010] Figure 1 ROBO1 expression. (A) Draft of a double layer of mammary alveoli including basal (myoepithelial and stem) cells (BC), luminal alveolar progenitor cells (AVP), and mammary alveoli (AV) cells. (B) Draft of SLIT / ROBO1. (C) RT-qPCR of Robo1 in primitive and PD18 luminal progenitor cells (LP), mature luminal cells (ML), and basal cells (BC) shows upregulation in luminal cells (LC) during pregnancy (n=3). (DG) Immunohistochemistry (D) or β-galactosidase (LacZ) staining (E) of ROBO1 in luminal cell subsets (arrows) of mature primitive ducts (D, E) and PD16 mammary alveoli (F). ROBO1 is also expressed in basal cells (arrows) of PD16 (F) and lactation day (LD3) (G) mammary alveoli. (SEM, t test p<0.01).

[0011] Figure 2 ROBO1 enhances mammary alveolar production. (A) Draft representation of the HC11 differentiation protocol. (B) Compared with Scr, KD (arrow) HC11 cells, dome formation was reduced in Robo1 cells 8 days after differentiation (Dif) in dexamethasone (1 μg / ml), insulin (5 μg / ml), and prolactin (Prl, 5 μg / ml) (DIP medium). 14Dome formation was rescued by Robo1 overexpression (o / e). Dome formation was negligible under no-dif conditions. RT-qPCR normalized relative to Scr control showed reductions in Robo1 and WAP after Robo1 knockdown (n=3). (C, D) Representative H&E-stained sections of intact PD18 Robo1 WT and KO mammaries (C) and contralateral transplanted growths (D), with graphs showing reduced alveolar area (10 images, n=3). (E) RT-qPCR showed decreased mammary gene expression in LD1 Robo1- / - mammaries relative to Robo1+ / + (n=3). (F) Representative immunohistochemistry and graphs of PD18 Robo1+ / + and Robo1- / - mammaries against WAP immunostaining (n=10 images, n=1). (G) Representative immunohistochemistry of PLIN2 and SMA in contralaterally transplanted Robo1 WT / KO growths on day 18 of gestation, with graphs showing decreased PLIN2 intensity in KO (10 images, n=3). (H) CUBIC method immunohistochemistry of ELF5 and CDH1 in contralaterally transplanted Robo1 WT / KO growths on day 18 of gestation, with graphs showing decreased ELF5 intensity (10 images, n=1). (I) Draft representation of the determination of milk production by monitoring pups' body weight. (J) Current data show a reduced gain in pups' body weight from Robo1- / - mothers (n=2). (SEM, **p<0.01, ***p<0.001).

[0012] Figure 3ROBO1 regulates NOTCH4 activation through direct interactions. (A) Genome browser snapshot of Robo1-regulated gene Hey1, with RNA-seq read coverage of Robo1+ / + and Robo1- / - cavitary progenitor (LP) cell samples plotted as a histogram (n=3). (B, C) RT-qPCR validation of Robo1-regulated gene expression showed that Notch effector genes were increased in Robo1 KO 1° mammary acinar progenitor (AVP) cells normalized relative to WT (B) (n=3) and in Robo1 knockdown HC11 cells normalized relative to control (Scr) cells (C) (n=3). (D) RT-qPCR showed that Notch effector genes Hey1 and Hes1 were significantly reduced in AVPs harvested from gestational age compared to primitive mammary glands (n=3). (E) GSI treatment rescued reduced dome formation in Robo1 knockdown (KD) HC11 cells (n=3). (F) GSI treatment (siR1+GSI) rescued reduced WAP and Lalba expression in Robo1 knockdown (siR1) HC11 cells. Notch4 knockdown (siN4) increased WAP and Lalba expression, as did dual knockdown of Notch4 and Robo1 (dKD) (n=1). (G) Robo1 reduced HC11 dome formation, a result that could be rescued by knockdown of Notch4 and dual knockdown of Robo1 / Notch4 (dKD) (n=2). (H) Endogenous ROBO1 was co-immunoprecipitated with NOTCH4, but not with NOTCH1 in MDA-MB-231 cell lysates (n=3). (IK) Cell-graded Western blots (I) and quantification showed increased NOTCH4 intracellular domain (N4-ICD) (J) and HES1 (K) in the nuclear fraction of Robo1 knockdown (siN4) differentiated HC11 cells. Robo1 overexpression (siR1+R1o / e) and GSI treatment (siR1+GSI) salvaged the effect (n=3). (L) Compared with undifferentiated (Undif) HC11 cells (n=1), differentiated (Dif) cells showed increased ROBO1 and pSTAT5, but decreased NOTCH4 intracellular domain (N4-ICD). (M) Endogenous NOTCH4 co-immunoprecipitated with ROBO1 from differentiated (Dif) and late-sensitized (-EGF) HC11 cells. SLIT did not appear to affect complex formation in differentiated HC11 cells, but a reduction in the complex was observed in late-sensitized (-EGF) cells. No ROBO1 / NOTCH4 complex precipitation was observed starting from early-sensitized cells (+EGF) or when control IgG was used (n=1). (SEM, *p<0.05, **p<0.01, ***p<0.001).

[0013] Figure 4 FACS-purified Robo1- / - AVP colonies were smaller and expressed little / no WAP: (A) Robo1- / - FACS-purified mammary alveolar progenitor cells (AVPs) cultured in Matrigel for 5 days with the Rho kinase inhibitor Y-27632 (10 μM, Tocris), Nrg1 (100 ng / ml, R&D), R-vertebral protein 1 (600 ng / ml, R&D), and prolactin (5 μg / ml, NHPP) were smaller than Robo1+ / + AVPs and expressed almost no WAP (n=2). (B) Compared to primary (1°) luminal epithelial cells Robo1+ / +, the level of NOTCH4 ICD (N4-ICD) in the nuclei of Robo1- / - cells was increased (n=2). (C) Draft representation of an assay testing Notch inhibition by treating Robo1+ / + and Robo1- / - mice with a γ-secretase (GSI) inhibitor. (D) Number of FACS-purified AVPs collected from Robo1+ / + and Robo1- / - mice treated with the mulcher or GSI. (n=3). (E) RT-qPCR of FACS-purified AVPs showed decreased Hey1 and Hes1 expression in Robo1+ / + mammary alveolar progenitor cells (AVPs), indicating that GSI inhibited Notch signaling. Compared to Robo1+ / + cells, Robo1- / - cells showed increased Hey1 and Hes1 expression and decreased Elf5 expression, an effect rescued by GSI treatment (n=1). (SEM, ***p<0.001).

[0014] Figure 5Robo2 inhibits alveolar production. (A) Robo2 knockdown increases dome formation and WAP expression three days after differentiation (arrows), a phenotype rescued by double knockdown of Robo1 and 2 (dKD). (B) Representative H&E-stained sections of DP16 Robo2 WT and KO mammaries. Quantification of alveoli in intact Robo2- / - mammaries and transplanted Robo2- / - growths shows increased alveolar area in Robo2- / - compared to Robo2+ / + tissues (n=10 images, n=1). (C) RT-qPCR of milk (top) and Notch effect (bottom) genes in Robo2- / - mammary glands (MG) normalized relative to Robo2+ / + controls. (D) β-galactosidase (LacZ) staining shows Robo2 in a subset of luminal cells (top) from DP16 mammary alveoli and a subset of basal cells (bottom) from ducts of retired breeders. (E) RT-qPCR of Robo2 and Robo1 in FACS-purified mammary epithelial cell subsets. Robo1 expression occurred in all populations, while Robo2 expression was limited to acinar progenitor cells (AVP) and basal (BC) cells. (F) Draft model showing ROBO2 inhibiting ROBO1, thereby allowing NOTCH4(N) activation to reduce acinar differentiation (left). ROBO1 extracellular domain (ECD) binds to ROBO2, thereby releasing ROBO1 and then inhibiting NOTCH4, and / or ECD binds and directly inhibits NOTCH4; both scenarios promote differentiation. Subplots showing ECDs for different constructs generated for the project (right). (G) Endogenous levels of ROBO2 and ROBO1 co-immunoprecipitation in HEK293 lysates and enhanced co-immunoprecipitation by SLIT2 / SLIT3 treatment, * is ROBO2, # is glycosylated ROBO2 (n=1). (SEM, n=3, *p<0.05).

[0015] Figure 6: ROBO1 extracellular domain. (A) Draft diagram of the domain structure showing the ROBO1 extracellular domain (ECD). (B) Western blot of ROBO1 ECD and control DCC ECD in lysates and conditioned medium from HEK cells overexpressing the plasmid construct. (C) Dot blot assay (left) and quantification (right) of ROBO1-Ig5 secreted from HEK cells overexpressing Robo-Ig5 in the absence and presence of heparin (300 ng / ml). Heparin moderately increases secretion (n=1). (D) Western blot of lysate titration in the absence and presence of heparin (300 ng / ml) shows no protein degradation. (E) HC11 dome formation assay shows that the function of ECD ROBO1-Ig2 and ROBO1-Ig5 is attenuated in the presence of heparin (n=1). (F) ECD binding assays using ROBO1-Ecto and ROBO2-overexpressing cells (top, green) and DCC-overexpressing cells (bottom, green). ROBO1-Ecto-HA (red) binds to ROBO2 but not to DCC.

[0016] Figure 7 : ROBO1 extracellular domain enhances differentiation. (A) Representative phase comparison of HC11 cells in the absence and presence of ROBO1-ECD treatment (top) and Bodipy 493 / 503 staining (bottom). (CG) Titration assays measuring the effect of ROBO1 ECD on HC11 differentiation showed that ROBO1-ECD increased dome formation in HC11 cells with increasing ECD concentration, but not with DCC-ECD (n=3 unless otherwise noted). (H) Titration assays measuring the effect of bovine ROBO1-Ig5 on HC11 differentiation showed increased dome formation with increasing concentration (n=2). (IK) RT-qPCR showed increased expression of WAP (n=1) and Lalba (n=2) genes after ROBO1-ECD treatment relative to the control. WAP (n=1) expression remained unchanged after DCC-Ig4 (0.7 μM) treatment. (LO)ROBO1 ECD titration showed that treatment with ROBO1-Ig5 (L, M) increased WAP (n=1) and PLIN2 (n=4) expression, and treatment with ROBO1-Ecto (N, O) increased WAP (n=3) and PLIN2 (n=2). (SEM, *p<0.05, **p<0.01, ***p<0.001)

[0017] Figure 8: The ROBO1 extracellular domain inhibits Notch activation. (A) RT-qPCR showed decreased Hey1 and Hes1 expression in HC11 cells treated with ROBO1-Ig5 and ROBO1-Ecto, but no decrease with ROBO1-Ig2 treatment (n=1). (B) Grading (cytoplasmic / nuclear) sensitized HC11 cells by Western blot analysis showed decreased HES1 and NOTCH4-ICD (N4-ICD) in the nuclear fraction with ROBO1-Ig5 treatment, with a decrease in the cytoplasmic fraction of NOTCH4-ICD (N4-ICD). (C) HC11 differentiation assays showed increased dome formation with ROBO1-Ig5 treatment under Scramble KD (Scr) conditions. Knockdown of Robo1 (shROBO1) reduced dome formation in untreated cells (control), an effect rescued by ROBO1-Ig5 treatment. Knockdown of Notch4 (shNotch4) increased dome formation in untreated cells (control), and this increase was not affected by ROBO1-Ig5 treatment (n=2).

[0018] Figure 9 Subcutaneous injection of the ROBO1 extracellular domain fragment increased branching: (A) WT primary murine mammary acinar progenitor cells (AVPs) were purified by FACS and grown in Matrigel in the absence of (control) and in the presence of ROBO1 ECD. All ROBO1 ECD fragments increased the number of organoids, with representative images showing AVPs grown in the presence of ROBO1-Ig5 (n=3). (B) WT primary bovine mammary acinar progenitor cells (AVPs) were purified by FACS and grown in Matrigel in the absence of (control) and in the presence of ROBO1-Ig5, which increased organoid size (n=2). (C) Draft representation of the ROBO1-Ig5 injection protocol. The ovaries (Ovx) of the animals were removed, then treated with hormones and injected with PBS or the ROBO1-Ig5 fragment. (D) Animals injected with ROBO1-Ig-5 showed increased mammary gland size and the number of first-degree (1°) branches (n=3). (E) The number of secondary / tertiary (2°, 3°) branches in the breast increased after ROBO1-Ig5 injection, but the branching density did not increase (n=3). (SEM, *p<0.05).

[0019] Figure 10: The ROBO1 extracellular domain fragment increases lobular mammary alveolar development. (A) Draft representation of the ROBO1 ECD-Fc fragment subcutaneous injection protocol. Robo1+ / + (WT) or Robo1- / - animals were subcutaneously injected with PBS or ROBO1 ECD-Fc on days 8.5, 11.5, and 14.5 of gestation (PD). Mammary glands were harvested at PD 17.5. (B, C) Representative H&E staining of simulated injection, WT, and Robo1- / - glands (control) shows the previously observed reduced lobular mammary alveolar development (arrows) and smaller, denser alveoli (asterisks) Robo1- / - phenotype. (D, E) Increased lobular mammary alveolar development and milk droplet production in WT (D) and Robo1- / - (E) glands subcutaneously injected with the ROBO1 ECD-Fc fragment. (F) Quantification of the percentage of mammary alveoli (%) showed a significant reduction in alveolar area in simulated Robo1- / - mammary tissue compared to control Robo1+ / + tissue, and a significant increase in alveolar area in either Robo1+ / + or Robo1- / - animals upon injection of the ROBO1 ECD-Fc (R1ECD) fragment. (SEM, *p<0.05, ***p<0.001).

[0020] Figure 11 The ROBO1 extracellular domain fragment increases milk production. (AC)RT-qPCR showed significantly reduced expression of WAP (A), XDH (B), and CSN2 (C) in simulated-injection Robo1- / - animals compared to Robo1+ / + animals. In Robo1+ / + animals injected with ROBO1ECD-Fc (R1ECD), WAP and CSN2 expression were significantly increased, and XDH showed an increasing trend. In Robo1- / - animals injected with ROBO1 ECD-Fc (R1ECD), WAP, XDH, and CSN2 expression were significantly increased. (DH)Immunohistochemistry (DG) and quantification (H) demonstrated significantly reduced milk protein expression in simulated-injection Robo1- / - mammary tissue compared to control Robo1+ / + tissue, and milk protein expression significantly increased with injection of the ROBO1 ECD-Fc (R1ECD) fragment into either Robo1+ / + or Robo1- / - animals. (SEM, *p<0.05, **p<0.01, ***p<0.001).

[0021] Figure 12Basal cells of the mammary gland require ROBO1 for alveolar differentiation and milk production. (AD) Chimeric organoids are generated by reconstructing purified luminal and basal cell populations. For WT tissue (GFP+ / +), ACTb-EGFP mice were used. (A) GFP+ / + basal cells and GFP+ / + luminal cells reconstructed as mammary organoids produce CSN2 (β-casein) after differentiation. (B) Robo1- / - basal cells and Robo1- / - luminal cells reconstructed as mammary organoids produce little / no CSN2 after differentiation. (C) Robo1- / - basal cells and GFP+ / + luminal cells reconstructed as mammary organoids produce little / no CSN2 after differentiation. (D) GFP+ / + basal cells and Robo1- / - luminal cells reconstructed as mammary organoids produce CSN2 after differentiation.

[0022] Figure 13 ROBO1 inhibits Jagged1 expression in basal cells. (A) Immunoblotting and quantification of HEK293 lysates from cells expressing increased levels of Robo1 plasmid (ROBO1) showed decreased JAGGED1 expression levels. GAPDH was a loading control. (B) Immunoblotting and quantification of HEK293 lysates from Robo1 knockdown (shRobo1) cells showed increased JAGGED1 expression while JAGGED2 expression remained unchanged. (C) Primary mammary epithelial cells were FACS purified from Robo1+ / + and Robo1- / - animals. JAGGED1 expression was increased in Robo1- / - basal cells compared to WT. (D) Immunohistochemistry of JAGGED1 and the basal marker cytokeratin 15 (CK14) showed increased JAGGED1 expression in Robo1- / - basal cells compared to Robo1+ / + mammary organoids. (***p<0.001).

[0023] sequence list

[0024] SEQ ID NO:1-Domestic Cow ROBO1-Ecto

[0025] SEQ ID NO:2-Domestic Cow ROBO1-Ig5

[0026] SEQ ID NO:3-Domestic Cattle-ROBO1-Ig2

[0027] SEQ ID NO:4-Homo sapiens ROBO1-Ecto

[0028] SEQ ID NO:5-Homo sapiens ROBO1-Ig5

[0029] SEQ ID NO:6-Homo sapiens ROBO1-Ig2

[0030] SEQ ID NO:7-BisonROBO1-Ecto

[0031] SEQ ID NO:8-Bison ROBO1-Ig5

[0032] SEQ ID NO:9-Bison ROBO1-Ig2

[0033] SEQ ID NO 10 - Bactrian Camel ROBO1 - Ecto

[0034] SEQ ID NO:11-Bactrian camel ROBO1-Ig5

[0035] SEQ ID NO:12-Bactrian camel ROBO1-Ig2

[0036] SEQ ID NO:13-Goat ROBO1-Ecto

[0037] SEQ ID NO:14-Goat ROBO1-Ig5

[0038] SEQ ID NO:15-Goat ROBO1-Ig2

[0039] SEQ ID NO:16-SheepROBO1-Ecto

[0040] SEQ ID NO:17-SheepROBO1-Ig5

[0041] SEQ ID NO:18-Sheep ROBO1-Ig2

[0042] SEQ ID NO:19-Wild Yak ROBO1-Ecto

[0043] SEQ ID NO:20-Wild Yak ROBO1-Ig5

[0044] SEQ ID NO:21-Wild Yak ROBO1-Ig2

[0045] SEQ ID NO:22-ROBOL1-Ecto

[0046] SEQ ID NO:23-Robo1-Ig5

[0047] SEQ ID NO:24-ROBO1-Ig2

[0048] SEQ ID NO:25-ROBOLI-Ecto

[0049] SEQ ID NO:26-Brown rat ROBO1-Ig5

[0050] SEQ ID NO:27-Brown rat ROBO1-Ig2

[0051] SEQ ID NO:28-Brown rat DCC Ig2

[0052] SEQ ID NO:29-Brown rat DCC Ig4

[0053] SEQ ID NO:30-Robo1 shRNA forward strand

[0054] SEQ ID NO:31-Robo1 shRNA reverse strand

[0055] SEQ ID NO:32-Notch4 shRNA forward strand

[0056] SEQ ID NO:33-Notch4 shRNA reverse strand

[0057] SEQ ID NO:34-Robo2 shRNA forward strand

[0058] SEQ ID NO:35-Robo2 shRNA reverse strand Detailed Implementation

[0059] Methods, agents, and compositions for promoting milk production in mammals are provided. Agents that can be used to promote milk production may contain agents that inhibit NOTCH4 activity. The agent may be a soluble ROBO1 extracellular domain, and the agent may inhibit NOTCH4 activity by binding to ROBO2 and / or by binding to NOTCH4. The agent may inhibit NOTCH4 by competing with ROBO1 for non-binding to ROBO2, thereby making ROBO1 available for inhibiting NOTCH4 activity. The agent may be an anti-NOTCH4 antibody that inhibits NOTCH4 activity. The agent may be an RNAi construct that inhibits NOTCH4 expression. The agent may be an RNAi construct that inhibits ROBO2 expression. This document also provides transgenic mammals that are genetically modified to express a soluble ROBO1 extracellular domain; inhibit ROBO2 expression; and / or inhibit NOTCH4 expression. Methods for promoting milk production in such transgenic mammals by administering one or more of the agents disclosed herein are also provided.

[0060] All disclosures and patents referenced in this specification are incorporated herein by reference as if each individual disclosure or patent were specifically and individually indicated to be incorporated herein by reference to disclose and describe the materials and / or methods relating to the cited disclosure. References to any publications are to their publication prior to the filing date and should not be construed as an admission that the methods, compositions, and transgenic mammals do not have prior rights to such publications, as the provided publication date may differ from the actual publication date, which may require independent verification.

[0061] It should be noted that, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the,” as used herein and in the appended claims, include plural references. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, the statements are intended to serve as a precondition for using exclusive terms such as “solely,” “only,” or negative limitations in conjunction with the elements of the claims.

[0062] As will be apparent to those skilled in the art, each of the individual aspects described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any other several aspects without departing from the scope or spirit of the method. Any method described may be performed in the order of the events described or in any other logically possible order.

[0063] definition

[0064] As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and binds to a target through at least one antigen-binding site. "Antibody" is used herein in the broadest sense and encompasses a wide variety of antibody structures, including but not limited to polyclonal antibodies, recombinant antibodies, monoclonal antibodies, chimeric antibodies (e.g., chimeras derived from antibody sequences of two or more different species such as humans, cattle, sheep, goats, and camels), humanized antibodies, human antibodies, bovine antibodies, sheep-derived antibodies, goat-derived antibodies, camel-derived antibodies, bispecific antibodies, multispecific antibodies, biantibodies, triantibodies, tetraantibodies, single-chain Fv (scFv) antibodies, single-domain antibodies (e.g., camel / llama antibodies), and antibody fragments.

[0065] The term "intact antibody" or "full-length antibody" refers to an antibody with a structure that is substantially similar to that of a natural antibody. It comprises an antibody consisting of two light chains and two heavy chains, each light chain including a variable region and a light chain constant region (CL), and each heavy chain including a variable region and at least heavy chain constant regions CH1, CH2, and CH3.

[0066] As used herein, the term "antibody fragment" refers to a molecule other than the complete antibody that comprises a portion of an antibody and typically includes an antigen-binding site. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, disulfide-linked Fv (sdFv), Fd, linear antibodies, single-chain antibody molecules (e.g., scFv), biantibodies, triantibodies, tetraantibodies, microantibodies, dual variable domain antibodies (DVD), single variable domain antibodies, and multispecific antibodies formed from antibody fragments.

[0067] As used herein, the term "variable region" refers to a region of the antibody light chain or antibody heavy chain involved in antibody-antigen binding. The variable regions of the antibody heavy and light chains have similar structures and typically include four framework regions and three complementarity-determining regions (CDRs) (also known as hypervariable regions).

[0068] The term "frame region" refers to amino acid residues other than CDR residues within the variable region. The variable region typically includes four frame regions: FR1, FR2, FR3, and FR4.

[0069] As used herein, the term "monoclonal antibody" refers to a substantially homogeneous group of antibodies that specifically recognize and bind to a single antigenic determinant or epitope. Individual antibodies comprising this group are identical, except for a small number of potentially naturally occurring mutations. The term "monoclonal antibody" encompasses full-length and complete monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) antibodies, fusion proteins comprising antibody fragments, and any other modified immunoglobulin molecules comprising antigen-binding sites. Furthermore, "monoclonal antibody" refers to such antibodies prepared using any number of techniques, including but not limited to hybridoma production, phage library display, recombinant expression, and transgenic animals.

[0070] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0071] As used herein, the term "humanized antibody" refers to a chimeric antibody that typically comprises a human immunoglobulin (e.g., a receptor antibody), in which native CDR residues are replaced by residues from a corresponding CDR derived from a non-human species such as a mouse, rat, rabbit, or non-human primate (e.g., a donor antibody), wherein the donor antibody possesses the desired specificity, affinity, and / or activity. In some cases, one or more residues within one or more framework regions of a human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may include residues not found in the receptor antibody or donor antibody. These modifications can be made to further improve and / or optimize antibody properties. Humanized antibodies may include a variable region containing all or substantially all of the CDRs corresponding to a non-human immunoglobulin and all or substantially all of the framework regions corresponding to a human immunoglobulin. In some aspects, humanized antibodies will include at least a portion of the immunoglobulin Fc region (e.g., the hinge region, CH1, CH2, and / or CH3), typically a portion of a human immunoglobulin. Similar definitions apply to bovine, sheep-like, goat-like, and camel-like antibodies.

[0072] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence corresponding to antibodies produced by humans and / or an antibody manufactured using any techniques known to those skilled in the art for manufacturing human antibodies. These techniques include, but are not limited to, phage display libraries, yeast display libraries, transgenic animals, and B-cell hybridoma techniques. Human antibodies as defined herein do not include humanized antibodies that include residues derived from non-human sources.

[0073] The terms “epitope” and “antigenic determinant” are used interchangeably herein and refer to the portion of an antigen or target that can be recognized and bound by a specific binder or conjugate (e.g., an antibody). When the antigen or target is a polypeptide, an epitope can be formed from both sequential amino acids and non-sequential amino acids juxtaposed through the ternary folding of a protein. Epitopes formed from sequential amino acids (also known as linear epitopes) are generally retained after protein denaturation, while epitopes formed through ternary folding (also known as conformational epitopes) are generally lost after protein denaturation. In a distinctive spatial conformation, an epitope typically contains at least 3, and more commonly at least 5, 6, 7, or 8–10 amino acids. Epitopes can be predicted using any of the numerous software bioinformatics tools available on the internet. X-ray crystallography can be used to characterize epitopes on target proteins by analyzing the amino acid residue interactions of antigen / antibody complexes.

[0074] As used herein, the term "specific binding" refers to a binder (e.g., an antibody) that interacts more frequently, more rapidly, for longer durations, with greater affinity, or in combination with a particular antigen, epitope, protein, or target molecule without interacting with alternative substances. Antibodies that specifically bind to antigens can be identified, for example, by immunoassay, ELISA, surface plasmon resonance (SPR) techniques (e.g., Biacore), FACS, or other techniques known to those skilled in the art.

[0075] The terms “polypeptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. The term “peptide” may be used to refer to polymers of fewer than 50 amino acids, for example, 5-50 amino acids. Polymers may be linear or branched, may include modified amino acids, and may be interrupted by non-amino acid components. The term also covers amino acid polymers that are naturally or through intervention; for example, through disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, but not limited to, non-natural amino acids) and other modifications known in the art. It should be understood that because some polypeptides in this disclosure may be antibody-based, the term “polypeptide” covers polypeptides as single-chain polypeptides and polypeptides with two or more related chains.

[0076] The terms “polynucleotide” and “nucleic acid” and “nucleic acid molecule” are used interchangeably herein and refer to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogues, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase.

[0077] In the context of two or more nucleic acids or peptides, the term "identical" or "percentage of identity" refers to the fact that two or more sequences or subsequences are identical or have a specified percentage of identical nucleotide or amino acid residues when compared and aligned against maximum correspondence (introducing vacancies if necessary), regardless of any conserved amino acid substitutions as part of sequence identity. The percentage of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain amino acid or nucleotide sequence alignments are well known in the art. These algorithms and software include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some respects, the two polynucleotides or peptides of this disclosure are substantially identical when compared and aligned against maximum correspondence, meaning that they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some respects, at least 95%, 96%, 97%, 98%, 99% of the nucleotide or amino acid residues of identity, as measured using sequence comparison algorithms or by visual inspection. In some aspects, identity exists in regions of sequences having a length of at least about 10, at least about 20, at least about 40-60 nucleotide or amino acid residues, at least about 60-80 nucleotide or amino acid residues, or any integer value therein. In some aspects, identity exists in longer regions exceeding 60-80 nucleotide or amino acid residues, such as at least about 80-100 nucleotide or amino acid residues, and in some aspects, the sequences being compared are substantially identical over their full length, said compared sequences being, for example, (i) coding regions of nucleotide sequences or (ii) amino acid sequences.

[0078] As used herein, the phrase "conservative amino acid substitution" refers to a substitution in which one amino acid residue is replaced by another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are broadly defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, phenylalanine substitution for tyrosine is considered a conserved substitution. Generally, conserved substitutions in peptide and / or antibody sequences do not eliminate the binding of the peptide or antibody to the target binding site. Methods for identifying conserved substitutions of nucleotides and amino acids that do not eliminate binding are well known in the art.

[0079] As used herein, the term "vector" means a construct capable of delivering and typically expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, and DNA or RNA expression vectors encapsulated in liposomes.

[0080] As used herein, the term "isolated" refers to polypeptides, peptides, soluble proteins, antibodies, polynucleotides, carriers, cells, or compositions in a form not found in nature. "Isolated" antibodies are substantially free of material from the cell source from which they originate. In some respects, isolated polypeptides, peptides, soluble proteins, antibodies, polynucleotides, carriers, cells, or compositions are those substances that have been purified to the point that they are no longer in their naturally occurring form. In some respects, isolated polypeptides, peptides, soluble proteins, antibodies, polynucleotides, carriers, cells, or compositions are substantially pure. Polypeptides, peptides, soluble proteins, antibodies, polynucleotides, carriers, cells, or compositions can be isolated from natural sources or from sources such as engineered cell lines.

[0081] As used herein, the term “substantially pure” means material that is at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0082] As used herein, the term "derived from" in the context of a polypeptide refers to a polypeptide having a sequence based on a protein from a specific source. A polypeptide derived from a protein from a specific source can be a variant of the protein from that specific source. For example, a polypeptide derived from a protein from a specific source can have a sequence modified relative to the sequence of the protein from which it originates. A polypeptide derived from a protein from a specific source shares at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with the protein from which it originates.

[0083] As used herein, the term “effective amount” refers to an amount of a drug (e.g., antibody, peptide, nucleic acid, etc.) sufficient to produce the desired effect in subjects such as mammals.

[0084] As used herein, references to “about” or “approximately” values ​​or parameters include (and describe) aspects relating to said value or parameter. For example, a description of “about X” includes a description of “X”.

[0085] As used in this disclosure and claims, unless the context clearly specifies otherwise, the singular forms “a”, “an”, and “the” include the plural forms.

[0086] In this document, the term "and / or" as used in phrases such as "A and / or B" is intended to encompass: both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0087] As used herein, the term RNAi construct encompasses RNA molecules and vectors whose presence within cells causes RNA interference (RNAi) and leads to reduced expression of transcripts targeted by the RNAi construct. The term includes siRNA, shRNA, and RNAi-inducible vectors.

[0088] As used herein, an RNAi-inducible vector is a vector in which the presence of the vector within a cell causes transcription of one or more RNA molecules that self-hybridize or hybridize with each other to form shRNA or siRNA. This term encompasses plasmids, such as DNA vectors or viral vectors. A vector may contain nucleic acids operatively linked to an expression signal that causes transcription of one or more RNA molecules that hybridize or self-hybridize to form siRNA or shRNA when the vector is present within a cell. Thus, the vector provides a template for the intracellular synthesis of one or more RNA molecules or their precursors.

[0089] Short interfering RNA (siRNA) comprises a double-stranded RNA of approximately 19 base pairs in length, and optionally further includes one or two single-stranded overhangs. siRNA can be formed from two hybridized RNA molecules, or alternatively from a single RNA molecule containing a self-hybridized portion. The double-stranded portion of the siRNA may contain one or more unpaired nucleotides. One strand of the siRNA contains a portion that hybridizes with a target transcript that is either perfectly complementary or has one, two, or two mismatches. In cases of non-perfect complementarity, any mismatch may be located at or near the siRNA terminus.

[0090] The term short hairpin RNA refers to an RNA molecule comprising at least two complementary moieties and at least one single-stranded moieties, wherein the at least two complementary moieties hybridize or are capable of hybridizing to form a double-stranded (double-stranded) structure (typically at least 19 base pairs in length) long enough to mediate RNAi, and the at least one single-stranded moieties form a loop, typically between about 1 and 10 nucleotides in length. The double-stranded moieties may, but typically do not, contain one or more protrusions consisting of one or more unpaired nucleotides.

[0091] This paper discloses an examination of the role of the ROBO receptor during mammary alveolar formation. Specifically, loss of Robo1 inhibits alveolar formation, while loss of Robo2 enhances it. Biochemical studies of cell lines reveal that ROBO1 specifically binds to NOTCH4 and inhibits NOTCH4 activation. ROBO1 was shown to be widely expressed throughout the mammary epithelial compartments, while ROBO2 expression was limited to alveolar progenitor cells and basal / myoepithelial cells (BCs). A fragment of the ROBO1 receptor, comprising a portion of the ROBO1 extracellular domain (ECD) that inhibits NOTCH4 signaling and promotes alveolar formation, is also disclosed. Alveolar formation is further disclosed to be enhanced in cells and mammals by treatment with antibodies that inhibit the binding of ROBO2 to ROBO1. Without being bound by theory, the findings disclosed herein demonstrate a deinhibitory circuit mechanism (ROBO2-|ROBO1-|NOTCH4) that regulates NOTCH4 signaling and thus the number of alveolar progenitor cells differentiating into lactating alveoli during each pregnancy.

[0092] Methods for enhancing milk production in mammals

[0093] This disclosure provides a method for promoting milk production in mammals. In some aspects, the method may comprise administering to the mammal a first agent that inhibits NOTCH4 activity, wherein the first agent is administered in an amount sufficient to inhibit NOTCH4 activity, thereby promoting milk production. The first agent may inhibit NOTCH4 activity by directly binding to the NOTCH4 protein, by inhibiting the binding of ROBO2 to ROBO1, by promoting the binding of ROBO1 to NOTCH4, by inhibiting the expression of NOTCH4, or by inhibiting the expression of ROBO2.

[0094] In some aspects, the first agent may include a soluble ROBO1 extracellular domain (ECD). In some aspects, the soluble ROBO1 ECD may contain the entire extracellular domain of ROBO1 or its ROBO2-binding fragment. In some aspects, the soluble ROBO1 ECD may contain at least two immunoglobulin (Ig) domains of ROBO1, for example, the first two Ig domains of ROBO1. In some aspects, the soluble ROBO1 ECD may contain at least five immunoglobulin domains of ROBO1. In some aspects, the soluble ROBO1 ECD may be derived from the extracellular domain of mouse, bovine, sheep, goat, or human ROBO1. In some aspects, the soluble ROBO1 ECD may contain an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in any one of SEQ ID NO:1-27. In some respects, the soluble ROBO1 ECD may comprise a sequence of any one of SEQ ID NO: 1-27 having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or up to 20) conserved amino acid substitutions. In some respects, the soluble ROBO1 ECD administered to mammals may be derived from a sequence of the ROBO1 protein expressed by mammals to reduce the immune response to the soluble ROBO1 ECD.

[0095] Soluble ROBO1 ECDs, which may contain the entire extracellular region of ROBO1 or its ROBO2-binding fragment, can be identified in any manner. For example, a soluble ROBO1 ECD that effectively inhibits NOTCH4 activity can be identified by performing an assay to measure the binding of the soluble ROBO1 ECD to ROBO2. The assay may include determining whether the soluble ROBO1 ECD binds to ROBO2 in the presence of a competing agent, such as full-length ROBO1 or a soluble ROBO1 ECD having the amino acid sequence described in any one of SEQ ID NO:1-27. In some aspects, a soluble ROBO1 ECD that effectively inhibits NOTCH4 activity can be identified by performing an assay to measure the binding of the soluble ROBO1 ECD to NOTCH4. The binding of the soluble ROBO1 ECD to ROBO2 and / or NOTCH4 can be measured by detecting the formation of the ROBO1 ECD::ROBO2 complex and / or the ROBO1 ECD::NOTCH4 complex. Other methods for identifying the binding of soluble ROBO1 ECD with ROBO2 and / or NOTCH4 can also be used.

[0096] In some respects, soluble ROBO1 is fused to or linked to a heterologous peptide. In some respects, the heterologous peptide is linked to the amino terminus, carboxyl terminus, or both of the two ends of the soluble ROBO1 ECD. As used herein, the term soluble in the context of ROBO1 ECD means that the ROBO1 ECD is not localized; the ECD described herein may and may not be localized to the cell surface because it lacks the transmembrane region required for cell surface localization. The soluble ROBO1 ECD also lacks the sequence of the intracellular region of ROBO1. In some respects, the soluble ROBO1 ECD peptide may be fused to an immunoglobulin Fc peptide (e.g., human IgG Fc, such as IgG1 Fc), serum albumin (e.g., human serum albumin, cynomolgus monkey serum albumin, or bovine serum albumin), or maltose-binding protein. In some respects, the soluble ROBO1 ECD may be fused to a protein tag that facilitates peptide purification or tracking. These protein tags include His tags, hemagglutinin tags, Fc regions (derived from Ig antibodies from humans, cattle, sheep, or goats, such as IgG, IgM, IgA, IgE, or IgD), or Myc tags.

[0097] In some respects, the first agent may be an anti-NOTCH4 antibody or a NOTCH4-binding fragment thereof that inhibits NOTCH4 activity. As used herein, unless the context clearly specifies otherwise, the term antibody encompasses its antigen-binding fragment. In some respects, antibodies include a variety of polyclonal antibodies that bind to different epitopes on an antigen. In some respects, antibodies are recombinant antibodies. In some respects, antibodies are monoclonal antibodies. In some respects, antibodies are chimeric antibodies. In some respects, antibodies are modified to provide reduced immunogenicity in mammals receiving the antibody. In some respects, antibodies are humanized antibodies. In some respects, antibodies are human antibodies. In some respects, antibodies are bovine antibodies. In some respects, antibodies are bovine antibodies. In some respects, antibodies are sheep-based antibodies. In some respects, antibodies are sheep antibodies. In some respects, antibodies are goat-based antibodies. In some respects, antibodies are goat antibodies. In some respects, antibodies are camel-based antibodies. In some respects, antibodies are camel antibodies. In some respects, antibodies are IgA, IgD, IgE, IgG, or IgM antibodies. In some respects, an antibody is an IgG antibody. In some respects, an antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some respects, an antibody is an antibody fragment including at least one antigen-binding site. In some respects, an antibody is a scFv. In some respects, an antibody is a disulfide-linked scFv. In some respects, an antibody is a Fab. In some respects, an antibody is a bispecific or multispecific antibody.

[0098] In some aspects, the first agent is a polyclonal antibody that binds to NOTCH4. Polyclonal antibodies can be prepared by any method known to those skilled in the art. In some aspects, polyclonal antibodies are produced by immunizing animals (e.g., cattle, sheep, camels, rabbits, rats, mice, goats, donkeys) with the antigen of interest (e.g., purified peptide fragments, recombinant proteins, or fusion proteins) through multiple subcutaneous or intraperitoneal injections. In some aspects, the antigen is conjugated to a carrier such as keyfora hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitors. The antigen (with or without the carrier protein) is diluted in sterile saline and typically combined with an adjuvant (e.g., complete or incomplete Freund's adjuvant) to form a stable emulsion. After a period of time, the polyclonal antibody is recovered from the immunized animal (e.g., from blood or ascites). In some respects, polyclonal antibodies are purified from serum or ascites according to standard methods in the art, including but not limited to affinity chromatography, ion exchange chromatography, gel electrophoresis, and / or dialysis.

[0099] In some aspects, the first agent is a monoclonal antibody that binds to NOTCH4. The monoclonal antibody can be prepared by any method known to those skilled in the art. In some aspects, the monoclonal antibody is prepared using hybridoma methods known to those skilled in the art. Immunization is performed on mice, rats, rabbits, hamsters, or other suitable host animals as described above. In some aspects, lymphocytes are immunized in vitro. In some aspects, the immunoantigen is a human protein or a fragment thereof. After immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol. Hybridoma cells are selected using specialized culture media known in the art, and unfused lymphocytes and myeloma cells do not survive the selection process. Hybridomas that produce monoclonal antibodies against the selected antigen can be identified by a variety of methods, including but not limited to immunoprecipitation, Western blotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore), and radioimmunoassay). After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, the clone can be subcloned by limiting dilution or other techniques. Hybridomas can be propagated in vitro using standard methods, or they can be propagated in animals as ascites tumors. Monoclonal antibodies can be purified from culture media or ascites fluid using standard methods in the art, including, but not limited to, affinity chromatography, ion exchange chromatography, gel electrophoresis, and dialysis.

[0100] In some aspects, monoclonal antibodies are prepared using recombinant DNA techniques known to those skilled in the art. For example, polynucleotides encoding the antibody are isolated from mature B cells or hybridoma cells using oligonucleotide primers that specifically amplify genes encoding the heavy and light chains of the antibody via RT-PCR, and their sequences are determined using standard techniques. The isolated polynucleotides encoding the heavy and light chains are then cloned into a suitable expression vector, which produces monoclonal antibodies when transfected into host cells that do not additionally produce immunoglobulins, such as *Escherichia coli*, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells.

[0101] In some respects, recombinant monoclonal antibodies are isolated from phage display libraries expressing variable domains or CDRs of the desired species (e.g., bovine or human). Screening of the phage library can be accomplished using a variety of techniques known in the art.

[0102] In some respects, monoclonal antibodies are modified using recombinant DNA technology to produce alternative antibodies. In some respects, the constant domains of the light and heavy chains of mouse monoclonal antibodies are replaced with constant regions of human, sheep, bovine, goat, or camel antibodies to produce chimeric antibodies. In some respects, constant regions are truncated or removed to produce the desired antibody fragment of the monoclonal antibody. In some respects, site-directed or high-density mutagenesis of variable regions is used to optimize the specificity and / or affinity of the monoclonal antibody.

[0103] In some respects, the anti-NOTCH4 antibody is a humanized antibody. Various methods for generating humanized antibodies are known in the art. In some respects, the humanized antibody comprises one or more amino acid residues introduced into its sequence from a non-human source. In some respects, humanization is performed by replacing one or more amino acids of the CDR sequence of a human antibody with corresponding amino acids from a non-human antibody (e.g., a mouse antibody). In some respects, the humanized antibody is constructed by replacing all six CDRs of the human antibody with corresponding amino acids from the CDRs of a non-human antibody (e.g., a mouse antibody).

[0104] The choice of which human heavy chain variable region and / or light chain variable region to use for generating humanized antibodies can be based on a variety of factors and through various methods known in the art. In some aspects, a "best-fit" approach is used when screening a whole library of non-human (e.g., rodent) antibody variable region sequences against known human variable region sequences. The human sequence most similar to the non-human (e.g., rodent) sequence is selected as the human variable region framework for the humanized antibody. In some aspects, a specific variable region framework derived from a common sequence of all human antibodies from a specific subgroup of the light or heavy chain is selected as the variable region framework. In some aspects, the variable region framework sequence is derived from the common sequence of the most abundant human subclass. In some aspects, human germline genes are used as the source of the variable region framework sequence.

[0105] In some respects, the anti-NOTCH4 antibody is a human antibody. Human antibodies can be prepared using various techniques known in the art. In some respects, the human antibody is produced by immortalized human B lymphocytes immunized in vitro. In some respects, the human antibody is produced by lymphocytes isolated from an immunized individual. Cells that produce antibodies against a target antigen can be produced and isolated under any circumstances. In some respects, the human antibody is selected from a phage library expressing human antibodies. Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable region gene lineages from unimmunized donors. Techniques for producing and using antibody phage libraries are well known in the art. After the antibody is identified, affinity maturation strategies known in the art, including but not limited to chain shuffling and site-directed mutagenesis, can be used to produce human antibodies with higher affinity. In some respects, the human antibody is produced in transgenic mice containing human immunoglobulin loci. After immunization, these mice are able to produce a complete human antibody library without producing endogenous immunoglobulins.

[0106] In some aspects, the antibody may be a bovine antibody or a whole bovine antibody. Methods for generating bovine antibodies from non-bovine antibodies may include forming a chimeric antibody that retains the CDR from the non-bovine antibody while other regions of the antibody may be replaced with corresponding sequences from the bovine antibody, introducing one or more amino acid residues from the bovine antibody into the chimeric antibody. In some aspects, a non-bovine antibody may be bovine-modified by replacing its constant region with a constant region from a bovine antibody. In some aspects, a non-bovine antibody may be bovine-modified by replacing its constant region with a constant region from a bovine antibody and its frame region with a frame region from a bovine antibody. In some aspects, bovine antibodies may be generated by replacing the CDRS of a bovine antibody with a CDR from a non-bovine antibody. In some cases, the antibody may be a whole bovine antibody generated using a gene sequence encoding a bovine antibody. Whole bovine antibodies may be generated in cattle, in bovine cell lines, in non-bovine cell lines genetically modified to express bovine antibodies, or in transgenic non-bovine animals genetically modified to express bovine antibodies. Similar methods may be used to generate species-specific antibodies that, when administered to a species, elicit a reduced immune response to the antibody. For example, in order to apply antibodies to sheep, goats, and camels respectively, sheep-like antibodies, goat-like antibodies, and camel-like antibodies can be produced.

[0107] Antibody sequence derivations (CDRs) are defined by those skilled in the art using various methods / systems. These systems and / or definitions have been developed and refined over many years and include Kabat, Chothia, IMGT, AbM, and Contact. The Kabat definition is based on sequence variability and is widely used. The Chothia definition is based on the location of structural loop regions. The IMGT system is based on sequence variability and location within the structure of variable domains. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on the analysis of available antibody crystal structures. An exemplary system is a combination of Kabat and Chothia. Software programs (e.g., abYsis) for antibody sequence analysis and CDR determination are available and known to those skilled in the art.

[0108] The specific CDR sequences defined herein are generally based on a combination of the definitions of Kabat and Chothia (exemplary system). However, it should be understood that references to one or more heavy chain CDRs and / or one or more light chain CDRs of a particular antibody will cover all CDR definitions known to those skilled in the art.

[0109] In some aspects, the anti-NOTCH4 antibody comprises an antibody in which at least one or more constant regions have been modified or deleted. In some aspects, the antibody may include modifications to one or more heavy chain constant regions (CH1, CH2, or CH3) and / or light chain constant regions (CL). In some aspects, the heavy chain constant regions of the modified antibody include at least one human constant region. In some aspects, the heavy chain constant regions of the modified antibody include more than one human constant region. In some aspects, modifications to the constant regions include the addition, deletion, or substitution of one or more amino acids in one or more regions. In some aspects, one or more regions are partially or completely deleted from the constant regions of the modified antibody. In some aspects, the entire CH2 domain has been removed from the antibody (ΔCH2 construct). In some aspects, the deleted constant region is replaced by a short amino acid spacer that provides some of the molecular flexibility typically conferred by a non-existent constant region. In some aspects, the modified antibody includes a CH3 domain directly fused to the hinge region of the antibody. In some respects, the modified antibody includes a peptide spacer inserted between the hinge region and the modified CH2 and / or CH3 domains.

[0110] It is known in the art that constant regions of antibodies mediate several effector functions, and these effector functions can vary depending on the antibody isotype. For example, the binding of the C1 component of complement to the Fc region of an IgG or IgM antibody (antigen binding) activates the complement system. Complement activation is important in opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and may be associated with autoimmune hypersensitivity. Additionally, the Fc region of an antibody can bind to cells expressing Fc receptors (FcRs). Many Fc receptors are specific to different classes of antibodies, including IgG (γ receptor), IgE (ε receptor), IgA (α receptor), and IgM (μ receptor). Antibody binding to Fc receptors on the cell surface triggers many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by cytotoxic cells (referred to as antibody-dependent cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

[0111] In some aspects, the anti-NOTCH4 antibody includes a variant Fc region. The amino acid sequences of the Fc regions of human IgG1, IgG2, IgG3, and IgG4 are known to those skilled in the art. In some aspects, the variant Fc region provides altered effector functions, which in turn affect the biological characteristics of the antibody. For example, in some aspects, the deletion or inactivation of the constant region (through point mutation or other means) reduces or eliminates the binding of the modified antibody to the Fc receptor during circulation. In some aspects, constant region modification increases the serum half-life of the antibody. In some aspects, constant region modification reduces the serum half-life of the antibody. In some aspects, constant region modification reduces, diminishes, or eliminates ADCC and / or complement-dependent cytotoxicity (CDC) of the antibody. In some aspects, substituting a specific amino acid in the human IgG1 Fc region with the corresponding IgG2 or IgG4 residue can reduce the effector function (e.g., ADCC and CDC) in the modified antibody. In some aspects, the antibody does not have one or more effector functions. In some aspects, the antibody has no ADCC activity and / or no CDC activity. In some respects, the antibody does not bind to Fc receptors and / or complement factors. In some respects, the antibody lacks effector function (e.g., "effectless" antibody). In some respects, constant region modifications increase or enhance the effector function of the antibody. In some respects, constant region modifications increase or enhance the antibody's ADCC and / or CDC. In some respects, the constant region is modified to eliminate disulfide bonds or oligosaccharide moieties. In some respects, the constant region is modified to add / replace one or more amino acids to provide attachment sites for one or more cytotoxins, oligosaccharides, or carbohydrates.

[0112] The constant regions of the antibodies described herein can be modified using well-known biochemical or molecular engineering techniques. In some aspects, antibody variants are prepared by introducing appropriate nucleotide changes into encoding DNA and / or by synthesizing the desired antibody or peptide. Using this technique, it is possible to disrupt the activity or effector function provided by a specific sequence or region while substantially maintaining the structure, binding activity, and other desired properties of the modified antibody.

[0113] This disclosure further includes additional variants and equivalents substantially homologous to the recombinant antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies or antibody fragments thereof described herein. In some aspects, it is desirable to enhance the binding affinity of the antibody. In some aspects, it is desirable to modulate the biological properties of the antibody, including, but not limited to, specificity, thermostability, expression level, effector function, glycosylation, immunogenicity, and / or solubility. Those skilled in the art will understand that amino acid changes can alter the post-translational processes of an antibody, such as changing the number or location of glycosylation sites, or altering membrane anchoring properties. Changes can be substitutions, deletions, or insertions of one or more nucleotides encoding the antibody or peptide, resulting in a change in the amino acid sequence relative to the native antibody or peptide sequence. In some aspects, amino acid substitution is the result of replacing one amino acid with another amino acid having similar structure and / or chemical properties, such as replacing leucine with serine, for example, conserved amino acid substitutions. The variant antibodies or peptides described herein can be produced using methods known in the art, including, but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

[0114] In some respects, agents that inhibit NOTCH4 activity, as described herein, are chemically modified. In some respects, soluble ROBO1 ECDs and / or anti-NOTCH4 antibodies have been chemically modified via glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, and / or linking to cellular ligands or other proteins. Any of these chemical modifications can be performed using known techniques.

[0115] In some aspects, the method may involve increasing milk production in a mammal species, including humans, cattle, sheep, goats, or camels, and the method may include administering to the mammal species a soluble ROBO1 ECD derived from human ROBO1, cattle ROBO1, sheep ROBO1, goat ROBO1, or camel ROBO1, respectively. In some aspects, the mammal is a female at a developmental stage suitable for milk production. For example, the mammal may be a female that has already developed mammary glands. In some aspects, the mammal is a female, cow, female deer, female sheep, or female camel. In some aspects, the mammal may become pregnant when the agent inhibiting NOTCH4 activity is administered to it. In some aspects, the mammal may have already given birth before the administration of the agent inhibiting NOTCH4 activity. For example, the mammal may give birth within 1-2 years of administration, such as within 3 months, 6 months, 1 year, or 18 months. In other aspects, the mammal is not pregnant. In some aspects, the mammal has not given birth before the administration of the agent inhibiting NOTCH4 activity. For example, mammals do not give birth within 1-2 years of administration, such as within 3 months, 6 months, 1 year, or 18 months.

[0116] In some aspects, agents for inhibiting NOTCH4 activity as described herein can be RNAi constructs that bind to NOTCH4 mRNA and reduce NOTCH4 expression. In some aspects, agents for inhibiting ROBO2 activity as described herein can be RNAi constructs that bind to ROBO2 mRNA and reduce ROBO2 expression. The RNAi construct can be a short interfering RNA (siRNA). The siRNA can be a short hairpin RNA (shRNA). The RNAi construct can be a microRNA (miRNA). Methods for preparing RNAi constructs to inhibit the expression of any known gene sequence are known to those skilled in the art. In some aspects, the siRNA for reducing NOTCH4 expression can comprise the nucleic acid sequence described in SEQ ID NO:32 or 33. In some aspects, the siRNA for reducing ROBO2 expression can comprise the nucleic acid sequence described in SEQ ID NO:34 or 35. In some aspects, the RNAi construct can be applied to mammals. In other aspects, nucleic acids...

[0117] In some aspects, methods for promoting milk production in mammals may involve administering one or more agents that inhibit NOTCH4 activity. In some aspects, the method may comprise administering at least one of a first agent and a second agent, wherein the first and second agents are independently selected from soluble ROBO1 ECD, an anti-NOTCH4 antibody, an RNAi construct that inhibits NOTCH4 expression, or an RNAi construct that inhibits ROBO2 expression. In some aspects, the method may comprise administering at least one of a first agent, a second agent, and a third agent, wherein the first, second, and third agents are independently selected from soluble ROBO1 ECD, an anti-NOTCH4 antibody, an RNAi construct that inhibits NOTCH4 expression, or an RNAi construct that inhibits ROBO2 expression. In some aspects, the method may include administering a first agent, a second agent, a third agent, and a fourth agent, wherein the first agent, the second agent, the third agent, and the fourth agent are independently selected from soluble ROBO1 ECD, an anti-NOTCH4 antibody, an RNAi construct that inhibits the expression of NOTCH4, or an RNAi construct that inhibits the expression of ROBO2.

[0118] One or more agents for inhibiting NOTCH4 activity can be administered to mammals via any suitable route to promote milk production, including parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implantation), intraperitoneal, intracisional, intra-articular, intraperitoneal, intracerebral (within brain parenchyma), and intraventricular), oral, nasal, vaginal, sublingual, intraocular, rectal, local (e.g., transdermal), sublingual, and inhalation. In some aspects, the one or more agents can be administered by direct injection, for example, into mammary tissue, such as intraductal injection.

[0119] Pharmaceutical agents and compositions thereof for inhibiting NOTCH4 activity

[0120] This document also provides pharmaceutical agents and compositions thereof that can be used to perform the methods disclosed herein.

[0121] In some aspects, peptides comprising the soluble ROBO1 ECD peptide as disclosed herein are provided. The soluble ROBO1 ECD peptide can be fused with heteropeptides as disclosed herein. In some aspects, nucleic acids encoding the soluble ROBO1 ECD peptide as disclosed herein are provided. A description of the soluble ROBO1 ECD peptide has been provided in previous sections and elsewhere herein and will not be repeated here for brevity. The soluble ROBO1 ECD can be produced using methods known in the art. The peptide can be produced wholly or partially using standard recombinant DNA techniques or chemical methods. Chemical methods for synthesizing the peptide can involve various solid-phase techniques that can be performed using automated peptide synthesizers (e.g., Biotage instruments). Chemical methods for synthesizing the peptide can involve the use of combinatorial methods. Additionally, the peptide can be modified using a variety of chemical methods known to those skilled in the art. Methods such as site-directed mutagenesis, alanine scanning, and / or PCR-based mutagenesis can also be used for peptide sequence alteration, substitution, and / or modification. Site-directed mutagenesis, cassette mutagenesis, restriction selection mutagenesis, and other techniques can be performed on cloned DNA to produce its soluble ROBO1 ECD, variants, fusions, chimeras, and other derivatives. A “generated” or “synthesized” polypeptide sequence is a polypeptide produced by any method involving artificial manipulation. Such methods include, but are not limited to, chemical synthesis, recombinant DNA technology, biochemical or enzymatic disruption of larger molecules, and combinations of the above methods.

[0122] When using recombinant technology to generate peptides such as soluble ROBO1 ECD peptides, peptides as intracellular or secreted proteins can be generated using any suitable construct and any suitable host cell, which can be prokaryotic or eukaryotic cells, such as bacterial (e.g., *Escherichia coli*) or yeast host cells. In some aspects, eukaryotic cells used as host cells for peptide generation include insect cells, mammalian cells, and / or plant cells. In some aspects, mammalian host cells are used and may include human cells (e.g., HeLa, 293, H9, and Jurkat cells); mouse cells (e.g., NIH3T3, L, and C127 cells); primate cells (e.g., Cos 1, Cos 7, and CV1); and hamster cells (e.g., Chinese hamster ovary (CHO) cells). In specific aspects, the peptides disclosed herein are generated in CHO cells or HEK cells. In some aspects, the peptides disclosed herein, such as soluble ROBO1ECD, are generated in cells cultured in the presence of heparin. For example, the culture medium may contain approximately 300 ng / ml of heparin. In other respects, the disclosed polypeptides, such as soluble ROBO1 ECD, are produced in cells cultured in a medium that does not contain significant amounts of heparin, for example, the medium may contain less than 300 ng / ml, 100 ng / ml, 50 ng / ml, 25 ng / ml, 10 ng / ml, or 1 ng / ml of heparin, or may not contain heparin at all.

[0123] Various host-vector systems suitable for expressing peptides can be employed according to standard procedures known in the art. See, for example, Sambrook et al., *Current Protocols in Molecular Biology*, 1989, Cold Spring Harbor Press, New York; and Ausubel et al., *Current Protocols in Molecular Biology*, 1995, edited by Wiley and Sons. Methods for introducing genetic material into host cells include, for example, transformation, electroporation, conjugation, calcium phosphate methods, etc. Transfer methods can be selected to provide stable expression of the introduced peptide-encoded nucleic acid. The peptide-encoded nucleic acid can be provided as a heritable addendum element (e.g., plasmid) or can be integrated into the genome. Various suitable vectors for generating the peptide of interest are commercially available.

[0124] Vectors can provide extrachromosomal maintenance or integration into the host cell genome. Expression vectors provide transcriptional and translational regulatory sequences and can provide inducible or constitutive expression, wherein coding regions are operatively linked under transcriptional control of transcription initiation and translation termination regions. Typically, transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. Promoters can be constitutive or inducible and can be strongly constitutive promoters (e.g., T7).

[0125] This document also provides nucleic acids encoding the peptides disclosed herein. In some aspects, the nucleic acid encoding the peptides disclosed herein is operatively linked to a promoter sequence that confers peptide expression. In some aspects, the nucleic acid sequence is a codon optimized for peptide expression in mammalian cells. In some aspects, the nucleic acid is deoxyribonucleic acid (DNA). In some aspects, the nucleic acid is ribonucleic acid (RNA). This document also provides vectors comprising nucleic acids encoding peptides for promoting milk production, as described herein. In some aspects, the vector is a viral vector.

[0126] In some respects, anti-NOTCH4 antibodies are provided as disclosed herein. Descriptions of anti-NOTCH4 antibodies have been provided in previous sections and elsewhere herein, and will not be repeated here for the sake of brevity. Anti-NOTCH4 antibodies for inhibiting NOTCH4 activity can be identified by any suitable method, such as assays and / or cell and animal models, as disclosed herein.

[0127] In some respects, RNAi constructs that inhibit NOTCH4 expression or ROBO2 expression, as disclosed herein, are provided. Descriptions of such RNAi constructs are provided in previous sections and elsewhere herein and will not be repeated here for the sake of brevity. RNAi constructs for inhibiting NOTCH4 activity can be identified by any suitable method, such as assays and / or cell and animal models, as disclosed herein.

[0128] This document also discloses pharmaceutical compositions comprising one or more NOTCH4 activity inhibitors as disclosed herein and pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable” means a substance approved or permitted by a regulatory agency or listed in the United States Pharmacopeia, the European Pharmacopeia, or other recognized pharmacopoeia for use in animals, including humans.

[0129] As used herein, the terms "pharmaceuticalally acceptable excipient, carrier, or adjuvant" or "acceptable pharmaceutical carrier" refer to an excipient, carrier, or adjuvant that can be administered to a subject in combination with at least one pharmaceutical agent without affecting the pharmacological activity of the agent. Typically, those skilled in the art and the U.S. FDA consider pharmaceutically acceptable excipients, carriers, or adjuvants to be inactive ingredients in any formulation.

[0130] As used herein, the terms "pharmaceutical formulation" or "pharmaceutical composition" refer to a formulation in which the biological activity of a pharmaceutical agent (e.g., an antibody) is exerted. Pharmaceutical formulations or compositions typically include additional components such as pharmaceutically acceptable excipients, carriers, adjuvants, buffers, etc.

[0131] In some respects, peptides and nucleic acids (e.g., encoding peptides or RNAi) are present in the pharmaceutical composition in therapeutically effective amounts. Therapeutically effective amounts can be determined based on observed effectiveness of the composition. Therapeuticly effective amounts can be determined using assays measuring the desired effect in cells, such as reporter cell lines whose expression of a reporter response to the peptides disclosed herein is regulated. The pharmaceutical composition can be administered ex vivo or in vivo to mammals to practice the methods and uses described herein.

[0132] The pharmaceutical compositions disclosed herein can be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are described herein. Suitable pharmaceutically acceptable or physiologically acceptable diluents, carriers, or excipients include, but are not limited to, nuclease inhibitors, protease inhibitors, suitable media such as physiological saline or citrate-buffered saline.

[0133] Solutions or suspensions intended for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents, such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetate, citrate, or phosphate; and agents for adjusting tension, such as sodium chloride or glucose. pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0134] Pharmaceutical compositions suitable for injectable applications typically comprise a sterile aqueous solution (in the case of water solubility) or dispersion, and a sterile powder for the ad hoc preparation of a sterile injectable solution or dispersion. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL... TM (BASF, Parsippony, New Jersey) or phosphate-buffered saline (PBS).

[0135] Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound, as needed, with one or a combination of the ingredients listed above into a suitable solvent, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing a base dispersion medium and other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which produce powders containing the active ingredient and any other desired components from their previously sterile filtered solutions.

[0136] Oral compositions typically contain an inert diluent or an edible carrier. For oral therapeutic administration, the active compound may be incorporated into excipients and used in the form of tablets, lozenges, or capsules, such as gelatin capsules. Oral compositions may also be prepared using liquid carriers used in mouthwashes. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following components or compounds with similar properties: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginate, Primogel, or corn starch; lubricants, such as magnesium stearate or sterotes; gliding agents, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylate, or orange flavorings. Formulations for oral delivery may be advantageously incorporated into the pharmaceutical preparation to improve stability in the gastrointestinal tract and / or enhance absorption.

[0137] For inhalation administration, the composition is formulated together with a delivery agent for delivery as an aerosol spray from a pressurized container or dispenser containing a suitable propellant (e.g., a gas such as carbon dioxide) or atomizer.

[0138] Systemic administration can also be performed via transmucosal or transdermal routes. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally well known in the art, and for transmucosal administration, they include, for example, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound and delivery agent are formulated into ointments, creams, gels, or lotions well known in the art. The composition can also be prepared as a suppository (e.g., using conventional suppository bases such as cocoa butter and other glycerides) or a retention enema for rectal administration.

[0139] On one hand, the composition is prepared together with a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation comprising an implant and a microencapsulated delivery system. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are readily apparent to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeting infected cells with monoclonal antibodies against antiviral antigens) can also be used as pharmaceutically acceptable carriers.

[0140] For ease of administration and dosage uniformity, oral or parenteral compositions may be formulated in unit dosage form. As used herein, unit dosage form refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit contains a predetermined amount of the active compound calculated to produce the desired effect, along with the required drug carrier.

[0141] As described above, nucleic acid molecules used as siRNA or shRNA transcription templates can be inserted into vectors that can be used as gene therapy vectors. Nucleic acid molecules encoding soluble ROBO1 ECD can also be inserted into vectors that can be used as gene therapy vectors. Typically, gene therapy vectors can be delivered to subjects by, for example, intravenous injection, local administration, or stereotactic injection. In some aspects, compositions comprising gene therapy vectors and delivery agents can be delivered orally or by inhalation and can be encapsulated or otherwise manipulated to protect them from degradation, etc. Pharmaceutical compositions comprising gene therapy vectors may contain an acceptable diluent or may include a sustained-release matrix in which a gene delivery medium is embedded. Alternatively, where the complete gene delivery vector can be generated intact from recombinant cells of, for example, retroviral or lentiviral vectors, the pharmaceutical article may contain one or more cells that generate the gene delivery system.

[0142] The pharmaceutical composition may be included in a container, package, or dispenser along with the instructions for use.

[0143] Genetically modified mammals

[0144] In some respects, transgenic mammals are provided that include gene modifications producing one or more of the following phenotypes: expression of a soluble ROBO1 extracellular domain; inhibition of ROBO2 expression; and inhibition of NOTCH4 expression. In some respects, the transgenic mammal may be a rodent, a cow, a sheep, a goat, or a camel.

[0145] In some respects, the phenotype is limited to breast tissue. In other respects, the expression of the phenotype is induced by using breast tissue-specific promoters, and the phenotype is limited to breast tissue.

[0146] In some respects, transgenic mammals can contain two gene modifications that produce the two phenotypes listed. In other respects, transgenic mammals can contain three gene modifications that produce all three phenotypes listed.

[0147] In some respects, methods for promoting milk production, as disclosed herein, may involve administering to a transgenic mammal at least one of a pharmaceutical composition that inhibits NOTCH4 activity, as disclosed herein.

[0148] In some respects, the transgenic animal may contain gene modifications that enable the expression of the soluble ROBO1 extracellular domain, and the method may further include administering to the transgenic animal a pharmaceutical composition comprising an RNAi construct including anti-ROBO1 antibody and anti-NOTCH4 antibody or an RNAi construct that inhibits the expression of ROBO2 and / or NOTCH4.

[0149] In some respects, the transgenic animal may contain gene modifications that inhibit the expression of ROBO2 and / or NOTCH4, and the method may further include administering a pharmaceutical composition comprising a soluble ROBO1 ECD as disclosed herein to the transgenic animal.

[0150] Transgenic mammals can be produced using methods known in the art. An exemplary method for preparing a transgenic mammal may include the following steps: 1) producing a gene construct containing a nucleic acid encoding a soluble ROBO1 ECD or a nucleic acid sequence transcribed into siRNA or shRNA targeting NOTCH4 or ROBO2 under the control of a promoter. The promoter may be a mammary gland-specific promoter or a broadly active promoter. 2) transfecting the gene construct into cells from a mammal, such as bovine cells, and selecting transgenic cells incorporating the gene construct. 3) (e.g., by applying an electrical pulse) fusing the transgenic cells with an enucleated oocyte from the same species as the transgenic cells (e.g., a bovine), and allowing the oocyte to develop into an embryo. 4) transplanting the embryo into a recipient mammal of the same species as the embryo (e.g., a bovine). 5) confirming that the embryo develops into a transgenic mammal.

[0151] experiment

[0152] ROBO1 is expressed in both the luminal and basal compartments and is upregulated during pregnancy:

[0153] Previous studies have focused on the role of SLIT / ROBO1 signaling in branching morphogenesis in primitive animals. 11,15,16To investigate the role of ROBO1 during pregnancy, the level of Robo1 mRNA in cells isolated from mammary glands was measured using RT-qPCR. Figure 1 C). Cells were collected from adult primitive mice and wild-type (WT) mice at day 18 of gestation (PD18). Figure 1 As indicated by C), and then purified into three subpopulations by fluorescence activated cell sorting (FACS): luminal progenitor cell subpopulation (LP, Lin-CD24). lo CD29 + CD61 + Mature luminal subgroup (ML, Lin-CD24) lo CD29 + CD61) and the basal subgroup (BC, Lin-CD24) + CD29 hi ) 17,18 The results showed that Robo1 was upregulated in luminal progenitor cells and mature luminal subsets, but not in the basal subset. Figure 1 C).

[0154] To assess the expression of ROBO1 and ROBO2 proteins in tissues, WT and Robo1 proteins from mice were analyzed. lacZ / + Immunohistochemistry was performed on tissue sections of mature primitive mammary glands. Figure 1 D) and β-galactosidase (lacZ) staining ( Figure 1 E). For day 16 of pregnancy (PD16) ( Figure 1 F) and day 3 of lactation (LD3) Figure 1 Immunohistochemistry and β-gal staining were also performed on breast sections of G. ROBO1 protein is expressed in the luminal cell subsets of mature primitive mammary glands and pregnant mammary glands. Figure 1 DF arrow). ROBO1 expression in the basal muscle epithelium was also observed in the mammary glands during pregnancy and lactation. Figure 1 (F, G arrows).

[0155] ROBO1 enhances alveolar production:

[0156] To investigate ROBO1 function during mammary alveolar development, Robo1 gene expression was suppressed in HC11 cells (Robo1 KD). HC11 cells are a mature prolactin-responsive lactation model. 19,20Cells in which Robo1 gene expression was not suppressed were referred to as WT or Robo1+ / + in this paper. To measure milk yield, cells were grown to confluence and then induced by treatment with epidermal growth factor (EGF, 10 ng / ml). EGF was administered in combination with carbon-stripped fetal bovine serum for three days, followed by one day of carbon-stripped fetal bovine serum in the absence of EGF. Cells were then treated for 3 to 5 days with a medium containing dexamethasone (1 μg / ml), insulin (5 μg / ml), and prolactin (PRl, 5 μg / ml) (DIP medium). Figure 2 A) to induce differentiation of these sensitized cells. Differentiation (Dif) leads to the development of a milk-filled dome. Figure 2 B). In response to treatment with DIP medium, a statistically significant reduction in milk dome formation and a statistically significant reduction in whey acid protein (WAP) gene expression were observed. Figure 2 B). If the cells are undifferentiated (Undif), there is almost no dome formation in WT or Robo1- / - cells ( Figure 2 B). Next, tissues from Robo1 knockout mice (Robo1- / -) and wild-type mice (WT or Robo1+ / +) were analyzed. Mammary glands were collected from WT and Robo1- / - animals at day 18 of gestation, and alveolar formation was analyzed by serial sectioning, carmine staining, and then quantification of the alveolar area located in sections at the top, middle, and bottom of the tissue. This analysis revealed a significant reduction in alveolar area in Robo1- / - mammary glands compared to WT mammary glands. Figure 2 C).

[0157] To ensure that this deficiency is due to Robo1 inhibition in mammary epithelial cells rather than a general lack of its potential impact on hormone production. 21 Tissues from Robo1- / - and littermate Robo1+ / + mice were transplanted contralaterally into hosts in which endogenous mammary epithelium had been pre-removed according to standard protocol. 22 Ten weeks later, the animals were mated and tissues were examined on day 18 of gestation. A significant reduction in alveolar area was observed in the transplanted Robo1- / -KO mammary glands. Figure 2 D), and the results were similar to those observed in the mammary glands of intact Robo1- / - animals. Figure 2 C). To assess the expression of specific markers regulated by pregnancy, Robo1- / - and Robo1+ / + tissues were collected on day 1 of lactation. RNA was extracted, and RT-qPCR was performed on genes known to be involved in milk production. Significantly decreased expression of WAP, whey protein α (Lalba), xanthine dehydrogenase (XDH), and lactolipoprotein (Btn1) was observed in Robo1- / - tissues. Figure 2 E).

[0158] The selected biomarkers were further evaluated using immunohistochemistry. WAP expression in the mammary glands of Robo1- / - and WT day 18 of pregnancy showed less WAP immunostaining in Robo1- / - tissues ( Figure 2 F). Immunostaining was performed on transplanted tissues from day 16 of pregnancy using an antibody specific to the lipid-binding protein perilipoplasmin 2 (PLIN2). Less PLIN2 immunostaining was observed in Robo1- / - breast tissue. Figure 2 G).

[0159] In addition, whole-organ tissue ablation is used to optimize tissue optical clarity and morphological preservation. Subsequently, a specific method for mammary alveolar production is employed. 6 Antibodies against the essential transcription factor ELF5 and double immunohistochemistry using antibodies specific to the cell-cell adhesion protein E-cadherin (CDH1) were employed. Figure 2 H). Compared to Robo1+ / + tissues, significantly less ELF5 staining was observed in Robo1- / - tissues (H). Figure 2 H).

[0160] The loss of Robo1 hinders milk production in the body:

[0161] To assess the effect of Robo1 expression on milk yield, hybridization was performed to produce heterozygous pups, which were then fed by Robo1- / - or WT females. Heterozygous pups were produced by crossing WT males with Robo1- / - females and by crossing Robo1- / - males with WT females. Litter sizes were limited to five pups, and these pups were weighed daily. Figure 2 I). The weight gain of heterozygous offspring fed by WT mothers was linear, while the weight gain of heterozygous offspring fed by Robo1- / - mothers was less ( Figure 2 J).

[0162] ROBO1 and NOTCH4 signal transduction interact and align for suppression:

[0163] Notch signaling is highly dose-sensitive, and the results depend on the level of receptor activity. 23 Upon ligand binding, the Notch receptor is activated via cleavage. This begins with extracellular cleavage, followed by γ-secretase-mediated intracellular cleavage that releases the Notch intracellular domain (ICD), which then enters the nucleus and regulates transcription. RNA sequencing analysis of FACS-purified subsets isolated from primitive mammary glands revealed higher expression of the Notch signaling effector Hey1 in the Robo1- / - luminal progenitor (LP) subset compared to Robo1+ / +. Figure 3 A).

[0164] Acinar progenitor cells (AVPs) were enriched from a FACS-purified luminal progenitor cell pool using the Sca / CD54 marker. Similar results were obtained from data derived from a large pool of luminal progenitor cells. Figure 3 A) RT-qPCR analysis of acinar progenitor cells (AVPs) revealed that Robo1- / - expression was significantly higher than Robo1+ / + expression in the three downstream Notch effectors (Hey1, Hes1, and Hey2). Figure 3 B). Similarly, in HC11 cells, the expression of Hey1, Hes1, and Hey2 was significantly higher than in WT cells after Robo1 expression was suppressed. Figure 3 C). Inhibition of Robo1 expression in HC11 cells also led to a significant decrease in the expression of the differentiation marker Elf5 relative to WT. Figure 3 C). These data indicate that inhibiting Robo1 expression in primary cells and tissue culture cells leads to upregulation of Notch effector genes and downregulation of the differentiation-promoting Elf5 gene, and further demonstrates that Notch signaling is activated in the absence of ROBO1.

[0165] Previous studies have shown that the formation of mammary alveoli requires downregulation of Notch signaling. 6 Especially Notch4 7-9 Acinar progenitor cells (AVPs) were purified by FACS from primitive animals (Virg) and animals on day 18 of gestation (PD18), and the expression of Notch4 target genes Hes1 and Hey1 was detected by RT-qPCR. Figure 3 D). It was observed that two Notch target genes were significantly downregulated in mammary alveolar progenitor cells isolated from the glands of pregnant animals compared with those isolated from the glands of primitive animals.

[0166] The role of Notch in HC11 cell differentiation assays was also evaluated. Inhibition of Robo1 expression (KD) resulted in significantly less HC11 nipple dome formation compared to control (Scr). Figure 3 E). HC11 cells with suppressed Robo1 expression (siR1) also showed lower WAP and Lalba expression compared to controls (Scr). Figure 3 F). Both effects were salvaged by treating cells with a γ-secretase inhibitor (GSI, RO4929097) (siR1+GSI). Figure 3 (E, F). This γ-secretase inhibitor can block Notch signaling, which further supports the idea that Robo1 loss enhances Notch4 signaling, thus producing an effect that can be salvaged by γ-secretase treatment.

[0167] In another experiment, Notch4 expression in HC11 cells (siN4) was suppressed. These cells showed greater WAP and Lalba expression compared to control cells (Scr). In yet another experiment, Robo1 and Notch4 expression in HC11 cells (dKD) were both suppressed, resulting in increased WAP and Lalba expression relative to control cells (Scr), similar to the WAP and Lalba expression levels observed with Robo1 inhibition plus GSI treatment (siR1+GSI). Figure 3 F). Notch4 knockdown resulted in a significant increase in the number of dome-shaped cells relative to control cells (Scr). Figure 3 G) — The results were consistent with higher expression in the milk gene. Figure 3 F). These data support the model of NOTCH4 inhibiting mammary alveolar formation. Inhibition of Robo1 (siR1) expression resulted in significantly fewer mammary domes compared to control cells (Scr). Figure 3 G), but simultaneously inhibiting Robo1 and Notch4 expression (dKD) resulted in the formation of more breast domes—the same effect observed when Notch4 expression (siN4) was inhibited alone. Figure 3 (G). In summary, these data suggest that ROBO1 and NOTCH4 play a role in regulating the same pathway of alveolar formation, with ROBO1 inhibiting NOTCH4 and NOTCH4 inhibiting alveolar formation.

[0168] Notch receptor activation can be regulated through direct interaction with binding partner. 24 Therefore, ROBO1 may bind to and directly inhibit the cleavage and activation of NOTCH4. To address this possibility, immunoprecipitation assays were performed using MBA-MD-231 cell lysates expressing detectable levels of all four Notch receptors (NOTCH1-4). Endogenous ROBO1 was immunoprecipitated with NOTCH4, but not NOTCH1, NOTCH2, or NOTCH3. Figure 3 H, and data not shown). Next, the expression and subcellular localization of the NOTCH4 intracellular domain (N4-ICD) and HES1 in control (Scr) and Robo1 (siR1) knockdown HC11 cells were examined. Robo1 expression was suppressed in HC11 cells, which were then prepared for differentiation as described above. Robo1 knockdown cells showed significantly higher expression of the nuclear NOTCH4 intracellular domain (N4-ICD) and HES1 compared to control cells (Scr). This effect was not observed in control Robo1 knockdown cells engineered to overexpress Robo1 (siR1+o / e) or in cells treated with the γ-secretase inhibitor GSI (siR1+GSI). Figure 3I-3K).

[0169] Further work addressed how to regulate the formation of the ROBO1 / NOTCH4 complex during the HC11 differentiation process. Expression analyses were performed during the HC11 differentiation phases (merging, initiation, and dome formation). 19,20 Western blot analysis of the intracellular domains of ROBO1, pSTAT5, and NOTCH4 during this period revealed higher levels of ROBO1(R1) and pSTAT5 compared to other stages. In contrast, the intracellular domain of NOTCH4 (N4-ICD) was expressed at lower levels during the dome formation stage. Figure 3 This finding is consistent with previous studies, suggesting that NOTCH4 signaling is attenuated during alveolar formation. 7-9 In both the presence and absence of SLIT2 and SLIT3, immunoprecipitation with anti-ROBO1 was used to downregulate NOTCH4, sensitization, and differentiation (Dif) of HC11 cells in the early (+EGF) and late (-EGF) stages. Figure 3 (M). ROBO1 / NOTCH4 complex formation appeared unaffected by SLIT2 / SLIT3 treatment in differentiated (Dif) HC11 cells. However, less ROBO1 / NOTCH4 complex formation was observed in the presence of SLIT2 and SLIT3 in late-stage sensitized cells (-EGF) compared to untreated cells. The ROBO1 / NOTCH4 complex was not detected in early-stage sensitized cells (+EGF) or in control IgG immunoprecipitates. In summary, these data suggest that ROBO1 directly binds to and inhibits NOTCH4 cleavage and signaling during mammary alveolar development, thereby hindering the differentiation of mammary epithelial cells into lactating cells.

[0170] ROBO1 inhibits Notch signaling in primary cells and mammals:

[0171] Because Robo1 loss enhances NOTCH4 signaling and inhibits HC11 cell differentiation, this process was further evaluated in primary cells and animals. Mammary acinar progenitor cells were purified by FACS, seeded at a single-cell density in Matrigel, and then grown for 5 days in medium supplemented with neuromodulatory proteins (100 ng / ml) and R-vertebral protein (42.5 ng / ml). The cells were then switched to DIP medium and allowed to differentiate for an additional 5 days. 25 Colonies growing from Robo1- / - mammary alveolar progenitor cells were observed to be smaller than those growing from WT mammary alveolar progenitor cells, and Robo1- / - colonies did not produce WAP ( Figure 4A). Immunostaining was performed on cultured WT and Robo1- / - primary coelomic cells. Significantly higher levels of the NOTCH4 intracellular domain (N4-ICD) were detected in the nuclei of Robo1- / - primary cells compared to WT cells. Figure 4 B). These studies indicate that Robo1- / - mammary alveolar progenitor cells (AVPs) contain high levels of the NOTCH4 intracellular domain in their nuclei and do not produce milk-producing organoids like their WT counterparts. This finding is consistent with the observed disruption of alveolar formation in Robo1- / - mammary glands. Figure 2 ).

[0172] In another study, Notch signaling was suppressed in an attempt to reverse the Robo1- / - phenotype. γ was selected as a previously successful in vivo inhibitor of mammary gland-related phenotypes. 29 Several different organs 26-28 Notch inhibitors. Mature primitive animals were treated with 10 mg / kg GSI or a mediator control for seven days. 29 ( Figure 4 C). After treatment, breast tissue was collected and analyzed by FACS and qPCR. It was observed that Robo1- / - breast tissue contained a greater number of amylopectin progenitor cells compared to the WT control. Figure 4 D). Treatment of Robo1- / - animals with a γ-secretase inhibitor resulted in these animals having the same number of mammary alveolar progenitor cells as WT animals. Figure 4 D). GSI treatment had no effect on the number of mammary alveolar progenitor cells in WT animals. Figure 4 D).

[0173] Examination of the expression of Notch effector genes (Hey1 and Hes1) revealed that GSI-treated animals showed lower expression of Notch effectors compared to those treated with the propagator. Figure 4 E). Although GSI inhibitors do not specifically target Notch receptors, this result suggests that the drug works in the mammary gland to reduce Notch signaling. Compared to AVPs from mate-treated WT animals, mate-treated Robo1- / - mammary acinar progenitor cells (AVPs) expressed higher levels of Hey1 and Hes1 (E). Figure 4 E). This result is similar to that observed in primary mammary alveolar progenitor cells and HC11 cells (E). Figure 3 B, C). Robo1- / - mammary acinar progenitor cells (AVPs) treated with the mulcher showed lower Elf5 expression levels than AVPs from mulcher-treated WT animals. Figure 4 E). This is related to the lower ELF5 expression level in Robo1- / - mammary glands compared to Robo1+ / + mammary glands (E). Figure 2H) and Robo1 knockdown cells had lower Elf5 levels than control (Scr) HC11 cells ( Figure 3 The observations were consistent with those in C). Further observations showed that, compared to media-treated Robo1- / - animals, GSI treatment reversed altered AVP gene expression in Robo1- / - animals—Hey1 and Hes1 expression of AVP was lower in GSI-treated Robo1- / - animals than in media-treated Robo1- / - animals, while Elf5 expression of AVP was higher in GSI-treated Robo1- / - animals than in media-treated Robo1- / - animals. Figure 4 E). In summary, this work demonstrates that ROBO1 restricts NOTCH4 signaling. In the absence of Robo1, NOTCH4 is activated—an effect pharmacologically inhibited by Notch signaling. Figure 4 Knockdown of E, 3E, 3F, 3I-K) or Notch4 gene expression ( Figure 3 F and G) reversed.

[0174] ROBO2 inhibits the production of mammary alveoli:

[0175] Inhibition of Robo2 in animals and cells resulted in a phenotype opposite to that produced by inhibition of Robo1 expression. In HC11 cells, inhibition of Robo2 expression (Robo2 KD) led to faster differentiation, higher WAP expression, and a larger number of mammary domes compared to control cells (Scr). Inhibition of both Robo1 and Robo2 in the same cell resulted in an indistinguishable number of mammary domes compared to the negative control. Figure 5 A).

[0176] Mammary alveolar formation was evaluated in both intact Robo2- / - mammary glands and contralateral Robo2- / - grafts. Significantly faster mammary alveolar formation was observed in both intact Robo2- / - mammary glands and Robo2- / - grafts compared to Robo2+ / + control mammary glands, as measured by alveolar area. Figure 5 B). Milk gene expression was higher in intact primitive Robo2- / - mammary glands (MG) than in Robo2+ / + controls, while Notch effector genes (Hey1, Hes1, and Hey2) were expressed lower in FACS-purified acinar progenitor cells (AVPs) from intact Robo2- / - mammary glands than in Robo2+ / + controls. Figure 5 C).

[0177] Robo2 expression in FACS-purified subsets of primitive mammary epithelial cells was assessed by RT-qPCR. Unlike Robo1, which is expressed in all subsets, Robo2 expression is more restricted—it is expressed at high levels in acinar progenitor cells (AVPs) and at lower levels in basal cells (BCs). Figure 5 D). Expression in duct progenitor cells (DP) was indistinguishable from that in mature lumen cells (ML); expression in ML was used for normalization. This was achieved by analyzing data from Robo2... lacZ / + β-galactosidase (lacZ) staining was performed on mammary gland sections to examine Robo2 expression in the tissue. The luminal cell subsets in the mammary alveoli on day 18 of pregnancy ( Figure 5 E, top) and basal localized cell subsets along the ducts of retired breeders ( Figure 5 Robo2 expression was observed in the E (bottom).

[0178] One interpretation of these phenotypic and expression data is that ROBO2 inhibits ROBO1 in mammary alveolar progenitor cells. During differentiation, ROBO2 is downregulated, releasing ROBO1, which then inhibits NOTCH4, thus forming a de-inhibition circuit (ROBO2-|ROBO1-|NOTCH4). Figure 5 (F left). In other words, inhibition of Robo2 expression allows ROBO1 to enhance alveolar differentiation. Inhibition of Robo1 expression allows NOTCH4 to inhibit alveolar differentiation.

[0179] The interaction between ROBO1 and ROBO2 is enhanced through SLIT:

[0180] The interaction between SLIT and ROBO proteins is evolutionarily conserved, as demonstrated by studies showing that human SLIT2 binds to Drosophila Robo1 with an affinity similar to its mammalian receptor, and vice versa. 30 Biochemical studies have shown that the interaction between this receptor / ligand pair involves the highly conserved second LRR domain of Slit and the conserved Ig1 domain of Robo, while the Ig2-Ig5 domains and all FN3 domains of ROBO1 appear to be unnecessary for binding. 31-34 Furthermore, research indicates that ROBO1 and ROBO2 can be expressed in cis... 32 35 ,36 and trans 37 The two interact with each other. This interaction also depends on the Ig domain. Recent crystallographic experiments have shown that unliganded ROBOs form compact homodimers in response to SLIT opening, thus allowing dimerization of dimers between ROBOs. 38.

[0181] The published model indicates that ROBO2 inhibits ROBO1. To investigate whether this inhibition is due to a direct interaction, an immunoprecipitation assay was performed on endogenous proteins in HEK cells using an antibody against ROBO1. A band that appeared to be glycosylated and bound by the ROBO2 antibody was immunoprecipitated with ROBO1. When immunoprecipitation was performed using cells with suppressed Robo1 expression, the intensity of this band was lower. Figure 5 G). Before lysate preparation and immunoprecipitation using anti-ROBO1 antibody, two strong ROBO2 bands were observed after cells were treated with SLIT2 and SLIT3 (1 μg each) for four hours. This indicates that SLIT2 and SLIT3 promote a more efficient interaction between ROBO1 and ROBO2. Figure 5 G).

[0182] ROBO1 receptor extracellular domain fragment binds to ROBO2:

[0183] The experiments disclosed in this paper demonstrate that ROBO1 and NOTCH4 form a complex that inhibits NOTCH4 activation, thus indicating a direct interaction between these two proteins. Previous studies have shown that soluble extracellular domain fragments of many transmembrane receptors can block homotropic and heterotropic interactions between transmembrane receptors, as well as interactions between transmembrane receptors and their ligands. 39 It can be hypothesized that the soluble ROBO1 extracellular domain (ECD) can similarly interfere with the interaction between ROBO1 and ROBO2. Constructs including the ROBO1 ECD can compete with endogenous ROBO1 for binding to ROBO2, thereby allowing endogenous ROBO1 to bind to NOTCH4 and inhibit NOTCH4 activation, thus enhancing alveolar differentiation and promoting milk production. Figure 5 F (right side). Soluble ROBO1 ECD can also directly bind to and inhibit NOTCH4 activation in a non-mutually exclusive manner, which also results in enhanced mammary alveolar differentiation. Figure 5 (F right side). Therefore, ROBO1 ECD can directly and indirectly inhibit NOTCH4 activation.

[0184] Three recombinant ROBO1 ECD constructs were constructed: one containing two immunoglobulin (Ig) domains (ROBO1-Ig2), another containing all five Ig domains (ROBO1-Ig5), and another containing the entire extracellular domain (ROBO1-Ecto). Figure 6 A). Among other constructs, HA (hemagglutinin), Myc, and human and mouse immunoglobulin Fc are fused with Robo1 ECD ( Figure 6A, 5F (right side). Extracellular domains missing in colorectal cancer (DCC) were constructed as negative controls. These extracellular domains are structurally similar to ROBO1 Ig superfamily members, comprising two Ig domains (DCC-Ig2) or four Ig domains (DCC-Ig4) and tagged with HA (…). Figure 6 A, 5F (right side). The expression and secretion of the construct were confirmed by overexpression of the construct in HEK293 cells and Western blot analysis of cell lysates and culture medium. Figure 6 B). Previous studies have shown that incubating cells with a highly sulfated variant of heparan sulfate enhances the secretion of some extracellular proteins. 40 Robo1-Ig5 was expressed in HEK-293 cells in the absence and presence of heparin (300 ng / ml) in this study. Culture medium was collected from these overexpressing cells on days 2, 4, and 6 post-plasmid transfection. A dot blot dilution assay was performed on the collected culture medium to assess the relative secretion of this soluble ROBO1 ECD. Figure 6 C). Soluble ROBO1-Ig5 secretion increases during this period, with heparin treatment showing a tendency to increase secretion. Figure 6 C). Culture medium samples from these ROBO1-Ig5 transfected cells were also subjected to TCA precipitation and analyzed by Western blotting, which showed intact ROBO1-Ig5 protein in the culture medium after 6 days in both the absence and presence of heparin. Figure 6 D).

[0185] Soluble ROBO1 ECD fragments ROBO1-Ig2 and ROBO1-Ig5, generated in the presence of heparin, were used for dome determination. Results showed that the soluble ROBO1 ECD fragments generated in the presence of heparin formed fewer domes than the same fragments generated in the absence of heparin. Figure 6 E). Because heparin treatment has only a moderate positive effect on ROBO1 ECD and has a detrimental effect on its function. Figure 6 Therefore, heparin was not used to generate soluble ROBO1 ECD fragments. The ability of the ROBO1 ECD fragment to bind to the ROBO2 receptor was tested by overexpressing Robo2 in Cos7 cells, treating cells with sodium azide to prevent protein internalization, and then incubating the cells with ROBO1-Ecto-HA 1H before fixation and immunostaining. The results showed that ROBO1-Ecto-HA bound to ROBO2, but DCC-Ig2-HA did not bind to ROBO2. Figure 6 G).

[0186] ROBO1 extracellular domain fragment enhances HC11 cell differentiation:

[0187] To determine whether the ROBO1 ECD fragment affected NOTCH4 signal transduction, phase contrast microscopy was used. Figure 7 A, B (top) and fluorescence microscopy using a hydrophobic Bodipy 493 / 503 bound to neutral lipids ( Figure 7 A and B) performed an HC11 assay to monitor dome formation. Undifferentiated (Undif) cells differed from those with interconnected protrusions visible in phase contrast and showed little / no Bodipy staining. Figure 7 A). Following differentiation and prolactin treatment, small lipid droplets accumulate and appear as dark-edged circles in phase contrast ( Figure 7 B, top), and appears as dotted green circles by Bodipy staining (B, top). Figure 7 B, bottom). Bodipy 493 / 503 staining revealed that treatment with ROBO1-Ecto resulted in a greater number of cells being completely surrounded by lipid droplets. Figure 7 B).

[0188] The dome number formation in response to ROBO1 ECD fragment titration was quantified. Higher concentrations of ROBO1-Ig2, ROBO1-Ig5, and ROBO-1-Ecto were significantly associated with higher dome formation rates. This result was not observed in responses to ECD fragments controlled with DCC-Ig2 or DCC-Ig4. Figure 7 CG). The bovine ROBO1-Ig5 construct was also tested in this assay, and, like the rat construct, formed more domes in response to treatment with higher concentrations of ROBO1-Ecto ECD. Figure 7 These studies collectively demonstrate that ROBO1-ECD promotes dome formation in HC11 cells.

[0189] To determine whether promoting dome formation also leads to higher milk yield, HC11 cells were differentiated with or without the ROBO1-ECD fragment, which was simultaneously added to the cells along with DIP medium. Cells were harvested and the expression of WAP and Lalba was assessed by RT-qPCR. Compared to the untreated control, treatment with different ROBO1-ECD resulted in a 6-9 fold increase in expression ( Figure 7 I, J). In cells treated with DCC-Ig4, WAP expression levels were not increased ( Figure 7 Next, Western blot analysis of WAP and PLIN2 was performed on cells treated with ROBO1-Ig5 and ROBO1-Ecto. An approximately two-fold increase in WAP and PLIN2 protein expression was observed in cells treated with ROBO-1ECD. Figure 7 LO).

[0190] ROBO1 extracellular domain fragments inhibit Notch signaling:

[0191] To examine the effect of the ROBO1-ECD fragment on Notch signaling, HC11 cells were treated with the ROBO1-ECD fragment and Notch effector expression was assessed. Treatment with ROBO1-Ig5 and ROBO1-Ecto resulted in lower expression of Hey1 and Hes1. Figure 8 A), although this effect was not observed in cells treated with ROBO1-Ig2 ( Figure 8 A). Additionally, HC11 cells were treated with ROBO-Ig5 during differentiation. These cells were then graded and Western blotted to detect HES1 and NOTCH4-ICD (…). Figure 8 B). Compared to the control treatment, treatment with ROBO1-Ig5 resulted in lower levels of HES1 and NOTCH4-ICD (N4-ICD) proteins in the nuclear fraction; ROBO1-Ig5 treatment also resulted in lower levels of NOTCH4-ICD in the cytoplasmic fraction. In summary, these results indicate that ROBO1-ECD inhibits Notch signaling.

[0192] This paper discloses a model in which the soluble ROBO1 ECD fragment binds to ROBO2, thereby preventing its binding to endogenous transmembrane ROBO1 and thus promoting the formation of a ROBO1 / NOTCH complex that interferes with Notch signaling. Figure 5 (F right). However, the ROBO1-ECD fragment may also directly bind to and inhibit NOTCH4. Therefore, to test whether the ROBO1-ECD fragment inhibits Notch in the absence of ROBO1, Robo1 expression in HC11 cells was suppressed. Cells lacking Robo1 expression were then treated with the ROBO1-ECD fragment, and their ability to form domes was assessed. As previously observed ( Figure 7 Treatment with the ROBO1-ECD fragment increased dome formation in control cells (Scr). Figure 8 C). Inhibition of Robo1 expression (shRobo1) also inhibited dome formation in control cells. Figure 8 C), as previously observed ( Figure 1 B, 3E). However, treatment of Robo1 expression-inhibited cells (shRobo1) with ROBO1-Ig5 resulted in the same dome formation level as control cells (Scr) treated with the ROBO1-ECD fragment. Figure 8C). Compared with control cells (Scr), inhibiting Notch4 (shNotch4) expression in the absence of the ROBO1-Ig5 fragment resulted in larger dome formation. Figure 8 C), as previously observed ( Figure 3 G). Treatment of these cells (shNotch4) with ROBO1-Ig5 resulted in the same dome formation level as untreated Notch4 knockdown cells (shNotch4). Figure 8 C). This indicates that in the absence of NOTCH4, ROBO1-Ig5 treatment does not further increase HC11 dome formation. In summary, the results show that NOTCH4 is a direct target of ROBO1-Ig5.

[0193] ROBO1 extracellular domain fragments enhance organoid formation and mammary branching:

[0194] The effects of the ROBO1 ECD fragment on the growth of primary mammary acinar progenitor cells (AVPs) in vitro and on branching morphogenesis in vivo were tested. FACS-purified mouse and bovine AVPs were seeded as single cells in Matrigel and grown for 10 days in both the absence and presence of the ROBO1-ECD fragment. Figure 9 A, B). Compared to the untreated control, treatment with ROBO1-ECD produced more mouse organs ( Figure 9 A). Compared to the untreated control, treatment with ROBO1-Ig5 produced larger bovine organs ( Figure 9 B). In vivo testing was also conducted by subcutaneous injection of the ROBO1-Ig5 fragment (7.5 mammary glands / kg / day) into ovariectomized animals orally administered the hormones in Nutella: estrogen (E, 1 μg / day), progesterone (P, 1 mg / day mammary gland / day), and prolactin (Pr1, 0.2 mg / day mammary gland / day). Figure 9 C). Breast tissue was harvested 14 days after ROBO1-Ig5 fragment treatment for carmine staining and evaluation. Compared to the untreated control, ROBO1-Ig5 treatment produced significantly larger areas and a greater number of first-degree (1°) branches. Figure 1 D). More secondary (2°) and tertiary (3°) branching was also observed in the glands; however, because the size of the glandular regions was larger in the more branched glands, the total branching density of the treated glands was not different from that of the control group. Figure 1 E). In summary, this study demonstrates that in vivo ROBO1-Ig5 treatment results in significantly more branching in the mammary gland. Other aspects may involve the ROBO1-ECD construct labeled with a mouse-Fc sequence. This tag is recognized by endogenous receptors that promote transport to tissues. 41 .

[0195] ROBO1 extracellular domain fragments increase mammary gland development and milk production in lobular mammary alveoli:

[0196] The effects of the ROBO1 ECD-Fc fragment on lobular mammary alveolar development during pregnancy were investigated. Three subcutaneous injections of the ROBO1 ECD fragment (7.5 mg / kg) were administered during pregnancy (PD 8.5, PD 11.5, and PD 14.5), compared with a control group receiving a simulant injection. Figure 10 A). Breast tissue was harvested at PD 17.5, and alveolar generation was analyzed by serial sectioning, hematoxylin and eosin (H&E) staining, and the area occupied by alveoli in sections located at the top, middle, and bottom of the tissue was then quantified. As previously observed, Robo1- / - alveolar area was significantly reduced compared to WT and simulated injection breast tissue, and Robo1- / - alveolar size was also reduced ( Figure 10 (B, C, F arrows, asterisk). Compared with the control group that mimicked the injection, injection of the ROBO1ECD-Fc fragment into WT and Robo1- / - animals resulted in a significant increase in alveolar area and alveoli filled with milk droplets.

[0197] To further evaluate milk yield, RT-qPCR was performed on the milk protein genes whey acid protein (WAP), xanthine dehydrogenase (XDH), and β-casein (CSN2). Compared with the control treatment, the expression of milk protein genes treated with ROBO1 ECD-Fc was significantly increased. Figure 11 AC). Milk expression was also assessed at the protein level via immunohistochemistry of tissue sections using an antibody against milk (#YNRM™, Accurate Chemical and Scientific Corp). Similarly, a significant increase in milk production was observed by injecting ROBO1 ECD into WT or Robo1- / - animals. Figure 11 These data collectively indicate that subcutaneous injection of the ROBO1 ECD fragment into pregnant animals increased lobular mammary alveolar development, milk protein genes, and milk yield.

[0198] basal cells of the mammary gland require ROBO1 for alveolar differentiation and milk production.

[0199] The mammary gland is a double-layered tissue composed of basal cells (basal compartment) and luminal cells (luminal compartment). Figure 1 A). ROBO1 expression was detected in both the mammary lumen and basal cells. Figure 1DG). To determine in which cell types ROBO1 functions to differentiate mammary alveolar progenitor cells into milk-producing mammary alveolar cells, chimeric organoids with ROBO1 expression were developed, such that cells including basal compartments or luminal compartments are composed of Robo1- / - cells (DG). Figure 12 C, D). As controls, WT and Robo1- / - organoids are produced by WT and KO cells, including basal and luminal compartments (WT / WT and KO / KO). Figure 12 A, B). For WT tissue (GFP+ / +), ACTb-EGFP mice were used to distinguish WT from KO cells. Organoids were produced by differential trypsin digestion to separate the two populations, followed by mixing the separated basal and luminal subpopulations to generate organoids (WT / WT, KO / KO, WT / KO, KO / WT), which were then cultured in Matrigel and subsequently differentiated for 5 days. WT / WT organoids, comprising both GFP+ / + basal cells and GFP+ / + luminal cells, formed large bilayer organoids that, upon differentiation, produced a lumen-filled latex (…). Figure 12 A). In contrast, KO / KO organoids, including Robo1- / - basal cells and Robo1- / - coelomic cells, produce smaller bilayered structures and produce little or no latex after differentiation. Figure 12 B). When Robo1- / - basal cells are mixed with WT coelenterate cells (KO / WT), the resulting chimeric organoids produce little / no milk after differentiation. Figure 12 C). However, when WT basal cells are mixed with Robo1- / - luminal cells (WT / KO), the resulting organoids produce milk ( Figure 12 D), similar to the milk production in WT / WT organoids ( Figure 12 A). These data suggest that in order for luminal cells to produce milk after hormone stimulation, ROBO1 needs to be expressed in the basal compartment of the mammary gland rather than the luminal compartment.

[0200] ROBO1 inhibits Jagged1 expression in basal cells:

[0201] One method for regulating Notch expression is to control the expression levels of Notch ligands Jagged1, Jagged2, or Delta. To investigate whether ROBO1 regulates Notch ligand expression, cells were transfected with a plasmid expressing an increased amount of Robo1. Forty-eight hours later, cells were harvested and immunoblotting was performed using antibodies against ROBO1, JAGGED1 (JAG1), and GAPDH (loading control). Figure 13A). Data showed that increased ROBO1 expression led to decreased JAGGED1 expression. Robo1 expression was then knocked down using siRNA. After 48 hours, JAGGED1 and JAGGED expression were assessed by Western blotting. Increased JAGGED1 expression was observed, while JAGGED2 expression remained unchanged. Figure 13 B, C). To examine whether this regulation of JAGGED1 occurs in vivo, primary WT and Robo1- / - mammary epithelial cell subsets were purified into basal, luminal, and stromal subsets using fluorescence activated cell sorting (FACS), and then JAGGED1 and cytokeratin 14 (CK14) were analyzed by Western blotting (sample control). Figure 13 D). More JAGGED1 was observed in Robo1- / - cells compared to Robo1+ / + basal cells. No detectable expression was observed in coelomic cells, and only moderate expression was observed in stromal cells; these data are similar to those obtained by knocking down Robo1 expression in cell lines. Figure 13 B). JAGGED1 expression was also assessed by immunostaining of Robo1+ / + and Robo1- / - organoids. Figure 13 E, F). More JAGGED1 expression was observed in Robo1- / - basal cells compared to Robo1+ / + organoids. In summary, these data indicate that ROBO1 inhibits JAGGED1 expression in mammary basal cells, and that loss of Robo1 leads to increased JAGGED1 expression. Increased JAGGED1 expression enhances NOTCH signaling in adjacent mammary alveolar progenitor cells, thereby inhibiting their differentiation into milk-producing mammary alveolar cells. Therefore, one mechanism by which ROBO1 regulates milk production is by controlling the level of the Notch ligand JAGGED1 in the basal compartment of the mammary gland.

[0202] Materials and Methods

[0203] animal:

[0204] All animal procedures were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of the University of California, Santa Cruz (UCSC). All Robo1 mice were bred and genotyped as described above. 11 .

[0205] Breast fat pad removal and transplantation:

[0206] Small mammary gland tissue fragments from 8-week-old WT and Robo1 KO mice were transplanted contralaterally into Foxn1 mice. nu The growth was collected from the pre-removed fat pad. On day 18.5 of gestation, the contralateral growth was collected and stained with carmine.

[0207] Whole breast carmine-alum assay:

[0208] Mouse mammary glands were surgically dissected, mounted on glass slides, and fixed in Carnoy's solution (25% glacial acetic acid and 75% ethanol). After brief dehydration, the glands were stained overnight in 0.2% carmine and 0.5% potassium aluminum sulfate, dehydrated in ethanol fractionation solutions (70%, 95%, and 100%), clarified in toluene, and mounted with mounting medium.

[0209] Analysis of fat pad filling:

[0210] Paraffin-embedded Robo1 KO or WT peritrichous tissue or contralateral growths were sectioned and stained with hematoxylin and eosin (H&E). Images were analyzed using ImageJ, and the percentage of fat pad filling was calculated by measuring the area occupied by the mammary alveoli.

[0211] Immunohistochemistry and β-galactosidase staining:

[0212] Tissues were fixed in 4% paraformaldehyde. Paraffin-embedded tissues were sectioned at 6 μm and serially loaded. Immunohistochemistry was performed according to standard protocols. For β-galactosidase staining, 40 mg / ml of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside was prepared in 1M phosphate buffer containing 1M MgCl2 and 10 mM potassium ferrocyanide. Frozen sections of tissue were treated with the prepared solution at 37°C for 1.5–24 hours, washed with PBS, dehydrated by ethanol, fixed with xylene, and covered with coverslips. 42 .

[0213] Microscopic examination:

[0214] Bright-field imaging was performed on a Biorevo BZ-9000 digital microscope (Keyence), and confocal microscopy was performed on a Nikon C2 confocal microscope and a Leica SP5 confocal microscope. The collected data were analyzed using ImageJ.

[0215] Immunoprecipitation:

[0216] Adhesive cells were lysed in 1 mL of 1X lysis buffer (137 nM NaCl, 10 mM Tris-HCl pH 8, 2 nM EDTA, 1 mM sodium orthovanadate), supplemented with 1% Igepal NP40 (Sigma), 1 mM benzoyl fluoride (PMSF), 1 mM leucopeptide, 1 mM aprotinin, and a phosphatase inhibitor (Roche Complete). Cell lysates were incubated with gentle agitation at 4°C for 15 min, followed by centrifugation at 12,000 rpm for 10 min. The soluble phase was incubated with 1 μg of antibody-conjugated dinoglobin A (Thermo-Fisher) at room temperature for 1 h or at 4°C for 4 h. Samples were washed and eluted according to protocol. The eluted protein complexes were mixed with 2X lysis buffer and incubated at 70°C for 10 min, followed by incubation at 100°C for 5 min.

[0217] Protein blot:

[0218] Protein lysates were prepared by directly lysing adherent cells in 1X sample buffer supplemented with 5% β-mercaptoethanol and boiling for 5 minutes. The protein lysates were analyzed by SDS-PAGE and transferred to PVDF at 400 mA for 90 minutes or overnight at 30 mA. Primary antibodies were used at the concentrations indicated in Table 1 and incubated overnight at 4°C. HRP-conjugated secondary antibodies (Jackson Labs) were used at a 1:3000 dilution and incubated at room temperature for 45 minutes. As previously described, all proteins were detected using the Clarity ECL on a BioRad Chemi-Doc MP imager (BioRad) and quantified using ImageLab software. 43 .

[0219] 2D cell cultures:

[0220] All cell lines were obtained from the American Type Culture Collection. MDA-MB-231 cells were cultured in DMEM growth medium (Gibco) supplemented with 10% heat-inactivated FBS (Seradigm) and 1X antibiotic antifungal agent (Gibco). Undifferentiated HC11 cells were cultured in RPMI-1640 growth medium (Gibco) supplemented with 10 μg / mL bovine insulin (Sigma-Aldrich) and 10 ng / mL human EGF (Peprotech). Primary LECs were collected from 8-week-old Robo1 KO or WT littermates, as previously described.15 .

[0221] 3D cell cultures:

[0222] FACS-purified AVPs were cultured in a matrix gel (BD Bioscience) at a density of 5000 cells / 100 μL and then cultured for 5 days in a basal medium (DMEM) supplemented with 100 ng / mL neuroregulatory protein (R&D), 42.5 ng / mL R-vertebral protein 1 (Peptec), F12 phenol-free medium, 10 mM HEPES, N2 (Gibco), and B27 (Gibco). To differentiate the AVPs, they were cultured in a medium supplemented with 10 mM HEPES, N2 (Gibco), and B27 (Gibco). -6 The acini were further grown for 5 days in a basal medium containing dexamethasone (Sigma-Aldrich), 10 μg / mL bovine insulin (Sigma-Aldrich), and 5 μg / mL prolactin (National Hormone and Peptide Program). 44 .

[0223] HC11 dome measurement:

[0224] HC11 cells were grown in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (BioFluid Technologies), 5 μg / mL insulin (Sigma), and 10 ng / mL epidermal growth factor (EGF; Sigma). To induce differentiation in HC11 cells, the confluence plates were continuously fed fresh medium (RPMI 1640 medium supplemented with 5% charred fetal bovine serum (BioFluid Technologies), 5 μg / mL insulin, and 10 ng / mL EGF) for 3 days, followed by initiation in RPMI 1640 medium supplemented with 5% charred fetal bovine serum (BioFluid Technologies) and 5 μg / mL insulin for 24 hours. After initiation, cells were injected every 24 hours into DIP medium (10% charred fetal bovine serum (BioFluid Technologies), 10% charred fetal bovine serum, and 10% ng / mL insulin). -6 Fresh medium was added to RPMI 1640 medium containing 5 μg / mL dexamethasone (Sigma-Aldrich), 5 μg / mL insulin, and 5 μg / mL prolactin (National Hormone and Peptide Program).

[0225] Lentiviral generation:

[0226] The generation of lentiviral particles for the scrambling Robo1, Robo2, and Notch4 knockdown experiments involved transfecting HEK293T cells with a combination of psPAX2, pMD2.G, and pLVTHM-scrambling-GFP (SCR) or pLVTHM-sh-target GFP. The filtered (0.45 μm) viral particles were then diluted in culture medium to infect target mammary lines (MDA-MB-231 and HC11 cells).

[0227] Isolation and flow cytometry of mammary epithelial cells:

[0228] As described, mechanically dissected groin and breast fat pads are prepared into cell suspensions for use in FACS. 17 As described, AVP was isolated using FITC-CD14 (clone Sa14-2; BioLegend) and ACP-Cy7-CD117 (clone 2B8; BioLegend). 14 .

[0229] In vivo γ-secretase inhibitors (GSI):

[0230] As described, the GSI inhibitor (RO4929097; MedchemExpress) was administered orally for 5 days at a dose of 10 mg / kg. 29 Mammary glands were collected 5 days after GSI or mediator treatment and prepared for single-cell analysis. Purified populations were collected and processed for RNA. FlowJo analysis was used to analyze the size of the purified populations.

[0231] RNA extraction and RT-qPCR:

[0232] Total RNA was collected from cells lysed in TRIzol reagent (Invitrogen) and the phase was separated from the overnight RNA precipitate in ethanol according to the manufacturer's protocol (Macias et al., 2011). RNA was further purified by treatment with TURBODNase (Ambion). Total RNA mass was analyzed by agarose gel electrophoresis and quantified using an ND-1000 spectrophotometer (NanoDrop). cDNA libraries were prepared from 1 μg of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories). Quantitative RT-PCR was performed in triplicate using a light Cycler 480SYBR Green I mixer (Roche) and quantified using the Bio-Rad Laboratories CFX'Connect real-time system and CFX Manager software (Bio-Rad Laboratories). Results were normalized to GAPDH.

[0233] ROBO1 extracellular domain generation:

[0234] To generate protein fragments, HEK cells were transfected with plasmids corresponding to the fragments of interest. PEI transfection was performed according to the Cytographica protocol. Twenty-four hours after transfection, the medium was replaced with OptiMEM. Eight days post-transfection, the medium was collected and centrifuged at 3000 x g for 10 minutes. The supernatant was then filtered through a 0.45 μm PVDF filter.

[0235] TCA precipitation:

[0236] Add 1 volume of TCA stock solution to 4 volumes of protein sample. Incubate at 4°C for 10 minutes. Centrifuge the tube at 14K rpm for 5 minutes. Remove the supernatant, keeping the protein precipitate intact, and wash the precipitate with 200 μl of cold acetone. Centrifuge at 14K rpm for 5 minutes. Repeat steps 4-6 for a total of 2 acetone washes. Dry the precipitate before resuspending it in sample buffer.

[0237] Bodipy 493 / 503 staining:

[0238] Place the cells in half a volume of buffer or culture medium. Prepare Invitrogen. TM BODIPY TM Prepare a 2X solution (2 μg / ml = 7.6 μM) of 493 / 503 dye in 0.5 mL of the same preheated buffer or culture medium (cell-free, BSA-free, or serum-free) and mix vigorously to mechanically emulsify the solution. Immediately add this solution to the cell solution, mix, and incubate for up to 30 minutes.

[0239] CUBIC immunofluorescence:

[0240] Glands were collected and fixed overnight at 4°C with 10% neutral buffered formalin (Sigma). The fixation was then quenched with PBST (0.1% Triton X-100; Sigma) containing 0.2% glycine (Fisher Scientific) for 2 × 10⁻⁶ min. The glands were then incubated in CUBIC Reagent 1A at 37°C for 48 h, followed by washing with PBST 3 × 10⁻⁶ min as described. 45The glands were blocked overnight at 4°C with PBST / 10% donkey serum (Sigma), and then incubated at 4°C for 48 hours with primary antibody in PBST / 5% donkey serum. The glands were then washed with PBST 3 × 1 hour and incubated at 4°C for 24 hours with secondary antibody diluted in PBST / 5% donkey serum. For counterstaining DNA, the glands were incubated with Hoechst diluted in PBST for 1 hour, followed by washing with PBST 2 × 1 hour. Finally, the glands were incubated with CUBIC reagent 2 at 4°C until they were removed, which typically takes 24 hours.

[0241] Intracatheter injection:

[0242] Preparation for Injection: Anesthetize mice using isoflurane chamber and applied eye lubricant. Continuously anesthetize mice with oxygen containing 2-4% isoflurane via a nasal cone. Remove hair from the nipple area using Nair chemical depilatories. Injection: On day 7 of gestation, bilaterally inject PBS or ROBO2 mAb into nipples #3, #4, and #5 of glands using a 33-gauge beveled needle (Hamilton) attached to a 50 μl syringe. The injection should be performed very slowly (approximately 40 μL / min) to minimize potential damage from rapidly flowing fluid within the ductal lumen. Post-Injection: Remove the animal from the nasal cone and transfer it to a separate cage for recovery. 46 .

[0243] Ovarian removal, hormone treatment, and subcutaneous injection:

[0244] Bilateral ovariectomy was performed on C57BL mice (8-10 weeks old), with a 1-week recovery period. 47 During the recovery period, mice were trained to take Nutella orally. Mice were administered Nutella in a mixture of 17β-estradiol (E, 1 μg, Sigma) and progesterone (P, 1 mg, Sigma) daily for three weeks. For ROBO1-Ig5 ECD, prolactin (Prl, 200 μg, NHPP) was administered orally via Nutella for one week (daily) starting from week 1 of E+P. For ROBO2 mAb, prolactin (Prl, 50 μg) was administered intraperitoneally for 2.5 weeks (daily) starting from week 1 of E+P. ROBO1-Ig5 ECD was administered subcutaneously over a two-week period starting from week 1 of E+P (7.5 mg / kg ROBO1-Ig5 ECD or PBS; daily). ROBO2 mAb or IgG isotype control mAb (250 μg / mouse) were administered subcutaneously twice weekly for 17 days starting 1 week after E+P. 48 .

[0245] Primers:

[0246]

[0247] Antibody:

[0248]

[0249] References:

[0250] 1. Macias, H. and Hinck, L. Mammary gland development. Wiley interdisciplinary reviews. Developmental Biology, 1, 533-557, doi:10.1002 / wdev.35 (2012).

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[0252] 3. Dontu, G. et al., Role of Notch signaling in cell-fate determination of human mammary stem / progenitor cells. Breast Cancer Research 6, R605-615, doi:10.1186 / bcr920 (2004).

[0253] 4. Bouras, T. et al., Notch signaling regulates mammary stem cell function and luminal cell-fate commitment. Cell Stem Cell, 3, 429-441, doi:10.1016 / j.stem.2008.08.001 (2008).

[0254] 5. Raouf, A. et al., Transcriptome analysis of the normal human mammary cell commitment and differentiation process. Cell Stem Cell, 3, 109-118, doi:10.1016 / j.stem.2008.05.018 (2008).

[0255] 6 Chakrabarti, R. et al., Elf5 regulates mammary gland stem / progenitor cell fate by influencing notch signaling. Stem Cells, 30, 1496-1508, doi:10.1002 / stem.1112 (2012).

[0256] 7 Jhappan, C. et al., Expression of an activated Notch-related int-3 transgene interferes with cell differentiation and induces neoplastic transformation in mammary and salivary glands. Genes and Development 6, 345-355 (1992).

[0257] 8. Gallahan, D. et al., Expression of a truncated Int3 gene in developing secretory mammary epithelium specifically retards lobular differentiation resulting in tumorigenesis. Cancer Research, 56, 1775-1785 (1996).

[0258] 9. Smith, GH et al. Constitutive expression of a truncated INT3 gene in mouse mammary epithelium impairs differentiation and functional development. Cell Growth Differ 6, 563-577 (1995).

[0259] 10 Marlow, R. et al., SLITs suppress tumor growth in vivo by silencing Sdf1 / Cxcr4 within breastepithelium. Cancer Research 68, 7819-7827, doi:10.1158 / 0008-5472.CAN-08-1357 (2008).

[0260] 11 Strickland, P., Shin, GC, Plump, A., Tessier-Lavigne, M. and Hinck, L., Slit2 and netrin1 act synergistically as adhesive cues to generate tubular bi-layers during ductal morphogenesis. Development 133, 823-832 (2006).

[0261] 12 Borrell, V. et al., Slit / Robo signaling modulates the proliferation of central nervous system progenitors. Neuron, 76, 338-352, doi:10.1016 / j.neuron.2012.08.003 (2012).

[0262] 13 Biteau, B. and Jasper, H., Slit / Robo signaling regulates cell fate decisions in the intestinalstem cell lineage of Drosophila. Cell Reports 7, 1867-1875, doi:10.1016 / j.celrep.2014.05.024 (2014).

[0263] 14 Shore, AN et al., Pregnancy-induced noncoding RNA (PINC) associates with polycomb repressive complex 2 and regulates mammary epithelial differentiation. PLoS Genet 8, e1002840, doi:10.1371 / journal.pgen.1002840 (2012).

[0264] 15. Macias, H. et al., SLIT / ROBO1 signaling suppresses mammary branching morphogenesis by limiting basal cell number. Dev Cell, 20, 827-840, doi:10.1016 / j.devcel.2011.05.012 (2011).

[0265] 16. Ballard, MS et al., Slit2 / Robo1 signaling regulates breast stem cell self-renewal via SNAI1 and mINSC. Cell Reports 13, 290-301, doi:10.1016 / j.celrep.2015.09.006 (2015).

[0266] 17 Shackleton, M. et al., Generation of a functional mammary gland from a single stem cell. Nature 439, 84-88, doi:10.1038 / nature04372 (2006).

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[0268] 19. Ball, RK, Friis, RR, Schoenenberger, CA, Doppler, W., and Groner, B., Prolactin regulation of beta-casein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial cell line. European Journal of Molecular Biology (EMBO J), 7, 2089-2095 (1988).

[0269] 20 Desrivieres, S. et al., Comparative proteomic analysis of proliferating and functionally differentiated mammary epithelial cells. Molecular Cell Proteomics, 2, 1039-1054, doi:10.1074 / mcp.M300032-MCP200 (2003).

[0270] 21 Dickinson, RE and Duncan, WC, SLIT-ROBO pathway: a regulator of cell function with implications for the reproductive system. Reproduction, 139, 697-704, doi:10.1530 / REP-10-0017 (2010).

[0271] 22 Deome, KB, Faulkin, LJ, Jr., Bern, HA, and Blair, PB, Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice. Cancer Research 19, 515-520 (1959).

[0272] 23 Andersson, ER, Sandberg, R. and Lendahl, U., Notch signaling: simplicity in design, versatility in function. Developmental Journal 138, 3593-3612, doi:10.1242 / dev.063610 (2011).

[0273] 24 Chillakuri, CR, Sheppard, D., Lea, SM, and Handford, PA, Notch receptor-ligand binding and activation: insights from molecular studies. Seminar on Cell and Developmental Biology, 23, 421-428, doi:10.1016 / j.semcdb.2012.01.009 (2012).

[0274] 25 Jarde, T. et al., Wnt and Neuregulin1 / ErbB signalling extends 3D culture of hormone-responsive mammary organoids. Nature Communications 7, 13207, doi:10.1038 / ncomms13207 (2016).

[0275] 26Bi,P. et al., Inhibition of Notch signaling promotes browning of white adipose tissue and ameliorates obesity. Nature Medicine, 20, 911-918, doi:10.1038 / nm.3615 (2014).

[0276] 27 Huang, R., Zhou, Q., Veeraragoo, P., Yu, H. and Xiao, Z., The Notch2 / Hes-1 pathway plays an important role in renal ischemia and reperfusion injury-associated inflammation and apoptosis, and the gamma-secretase inhibitor DAPT has a nephroprotective effect. Renal Failure, 33, 207-216, doi:10.3109 / 0886022X.2011.553979 (2011).

[0277] 28 Chen, Y. et al., Inhibition of Notch signaling by a gamma-secretase inhibitor attenuate shepatic fibrosis in rats. PLOS ONE, 7, e46512, doi:10.1371 / journal.pone.0046512 (2012).

[0278] 29 Regan, JL et al., Aurora A kinase regulates mammary epithelial cell fate by determining mitotic spindle orientation in a Notch-dependent manner. Cell Reports 4, 110-123, doi:10.1016 / j.celrep.2013.05.044 (2013).

[0279] 30 Brose, K. et al., Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell, 96, 795-806 (1999).

[0280] 31Howitt, JA, Clout, NJ, and Hohenester, E., Binding site for Robo receptors revealed by dissection of the leucine-rich repeat region of Slit. European Journal of Molecular Biology 23, 4406-4412, doi:10.1038 / sj.emboj.7600446 (2004).

[0281] 32 Liu, Z. et al., Extracellular Ig domains 1 and 2 of Robo are important for ligand (Slit) binding. Molecular Cell Neurosci 26, 232-240, doi:10.1016 / j.mcn.2004.01.002 (2004).

[0282] 33 Morlot, C. et al., Structural insights into the Slit-Robo complex, Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci USA) 104, 14923-14928, doi:10.1073 / pnas.0705310104 (2007).

[0283] 34 Fukuhara, N., Howitt, JA, Hussain, SA, and Hohenester, E., Structural and functional analysis of slit and heparin binding to immunoglobulin-like domains 1 and 2 of Drosophila Robo. Journal of Biol Chem. 283, 16226-16234, doi:10.1074 / jbc.M800688200 (2008).

[0284] 35 Hivert, B., Liu, Z., Chuang, CY, Doherty, P., and Sundaresan, V., Robo1 and Robo2 are homophilic binding molecules that promote axonal growth. Molecular and Cellular Neuroscience 21, 534-545 (2002).

[0285] 36 Evans,TA and Bashaw,GJ, Robo receptor immunoglobulin domains promote distinct axon guidance decisions (Current Biology, 20, 567-572, doi:10.1016 / j.cub.2010.02.021 (2010)).

[0286] 37 Evans, TA, Santiago, C., Arbeille, E., and Bashaw, GJ. Robo2 acts in trans to inhibit Slit-Robo1 repulsion in pre-crossing commissural axons. ELife, 4, e08407, doi:10.7554 / eLife.08407 (2015).

[0287] 38 Aleksandrova, N. et al., Robo1 Forms a Compact Dimer-of-Dimers Assembly. Structure, 26, 320-328e324, doi:10.1016 / j.str.2017.12.003 (2018).

[0288] 39 Peschon, JJ et al., An essential role for ectodomain shedding in mammalian development. Science, 282, 1281-1284 (1998).

[0289] 40 Lupu, C. et al., Cellular effects of heparin on the production and release of tissue factor pathway inhibitor in human endothelial cells in culture. Arterioscler, Thrombus and Vascular Biology, 19, 2251-2262 (1999).

[0290] 41 Richter, WF and Jacobsen, B., Subcutaneous absorption of biotherapeutics: knowns and unknowns. Drug Metabolism and Disposal 42, 1881-1889, doi:10.1124 / dmd.114.059238 (2014).

[0291] 42 Brisen, C. et al., Prolactin controls mammary gland development via direct and indirect mechanisms. Developmental Biology, 210, 96-106. (1999).

[0292] 43 Le, LT et al., Loss of miR-203 regulates cell shape and matrix adhesion through ROBO1 / Rac / FAK in response to stiffness. Journal of Cell Biology, 212, 707-719, doi:10.1083 / jcb.201507054 (2016).

[0293] 44 Harburg, G. et al., SLIT / ROBO2 signaling promotes mammary stem cell senescence by inhibiting Wnt signaling. Stem Cell Reports, 3, 385-393, doi:10.1016 / j.stemcr.2014.07.007 (2014).

[0294] 45 Lloyd-Lewis, B. et al., Imaging the mammary gland and mammary tumors in 3D: optical tissue clearing and immunofluorescence methods. Breast Cancer Research, 18, 127, doi:10.1186 / s13058-016-0754-9 (2016).

[0295] 46 Krause, S., Brock, A. and Ingber, DE, Intraductal injection for localized drug delivery to the mouse mammary gland, Journal of Visual Experimentation, doi:10.3791 / 50692 (2013).

[0296] 47Strom, JO, Theodorsson, A., Ingberg, E., Isaksson, IM and Theodorsson, E., Ovariectomy and 17β-estradiol replacement in rats and mice: a visual demonstration. Journal of Visual Experimentation, e4013, doi:10.3791 / 4013 (2012).

[0297] 48Machholz, E., Mulder, G., Ruiz, C., Corning, BF., and Pritchett-Corning, KR., Manual restraint and common compound administration routes in mice and rats. Journal of Visual Experimentation, doi:10.3791 / 2771 (2012).

[0298] Although preferred aspects of the invention have been shown and described herein, it will be apparent to those skilled in the art that such aspects are provided by way of example only. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the aspects of the invention described herein can be used to practice the invention. The following claims are intended to define the scope of the invention and thus cover the methods and structures within the scope of these claims and their equivalents.

[0299] For the sake of completeness, certain aspects of the peptides, compositions, and methods disclosed herein are set forth in the following numbered clauses:

[0300] 1. A method for promoting milk production in mammals, the method comprising:

[0301] The first agent that inhibits NOTCH4 activity was administered to the mammal in an amount sufficient to inhibit NOTCH4 activity, thereby promoting milk production.

[0302] 2. The method according to Clause 1, wherein the first agent inhibits NOTCH4 activity by directly binding to the NOTCH4 protein, by inhibiting the binding of ROBO2 to ROBO1, by promoting the binding of ROBO1 to NOTCH4, by inhibiting the expression of NOTCH4, or by inhibiting the expression of ROBO2.

[0303] 3. The method according to Clause 1, wherein the first agent comprises a soluble ROBO1 extracellular domain (ECD).

[0304] 4. The method according to Clause 3, wherein the soluble ROBO1 ECD is a rodent, cow, sheep, goat, camel or human ROBO1 ECD.

[0305] 5. The method according to clause 3 or 4, wherein the ROBO1 ECD comprises a heterologous polypeptide.

[0306] 6. The method according to Clause 5, wherein the heterologous polypeptide includes a His tag, a hemagglutinin tag, an immunoglobulin (Ig) Fc region, or a Myc tag.

[0307] 7. The method according to Clause 1, wherein the first agent comprises an RNAi construct that inhibits the expression of NOTCH4 or ROBO2.

[0308] 8. The method according to Clause 7, wherein the RNAi construct is a short interfering RNA.

[0309] 9. The method according to Clause 1, wherein the first agent comprises an anti-NOTCH4 antibody or a NOTCH4-binding fragment thereof.

[0310] 10. The method according to Clause 9, wherein the first agent comprises multiple polyclonal anti-NOTCH4 antibodies.

[0311] 11. The method according to Clause 9, wherein the anti-NOTCH4 antibody or its NOTCH4-binding fragment is a monoclonal antibody or its NOTCH4-binding fragment.

[0312] 12. The method according to Clause 10, wherein the polyclonal anti-NOTCH4 antibody is a mouse, bovine, sheep, goat, camel, or human polyclonal antibody, and wherein the species producing the polyclonal antibody matches the species of the mammal to which the first agent is administered.

[0313] 13. The method according to Clause 11, wherein the monoclonal antibody or its NOTCH4 binding fragment is a bovine, sheep, goat, or human monoclonal antibody or its NOTCH4 binding fragment, and wherein the species from which the monoclonal antibody is derived matches the species of the mammal to which the first agent is administered.

[0314] 14. The method according to Clause 13, wherein the anti-NOTCH4 monoclonal antibody or its NOTCH4 binding fragment is bovine, sheep-derived, goat-derived, camel-derived, or humanized.

[0315] 15. The method according to Clause 1, wherein the first agent comprises a soluble ROBO1 extracellular domain, the method further comprising administering a second agent that inhibits NOTCH4 activity to the mammal in an amount sufficient to inhibit NOTCH4 activity.

[0316] 16. The method according to Clause 15, wherein the second agent comprises an RNAi construct that inhibits the expression of NOTCH4 or ROBO2.

[0317] 17. The method according to Clause 16, further comprising a third agent comprising an RNAi construct that inhibits the expression of NOTCH4 or ROBO2.

[0318] 18. The method according to Clause 1, wherein the method comprises administering at least one of a first agent, a second agent, a third agent, and a fourth agent that inhibits NOTCH4 activity, wherein each of the first agent, the second agent, the third agent, and the fourth agent is independently selected from soluble ROBO1 ECD, an anti-NOTCH4 antibody, an RNAi construct that inhibits NOTCH4 expression, and an RNAi construct that inhibits ROBO2 expression.

[0319] 19. A polypeptide comprising:

[0320] Soluble ROBO1 extracellular domain fused with a heterologous peptide.

[0321] 20. The polypeptide according to Clause 19, wherein the soluble ROBO1 ECD is a rodent, bovine, sheep, goat, or human ROBO1 ECD.

[0322] 21. The polypeptide according to Clause 20, wherein the heterologous polypeptide includes a His tag, a hemagglutinin tag, a human or mouse Fc region, a Myc tag, or a fluorescent protein.

[0323] 22. A pharmaceutical composition comprising:

[0324] The polypeptide and pharmaceutically acceptable carrier as described in any one of Clauses 19 to 21.

[0325] 23. The pharmaceutical composition described in Clause 22, for promoting milk production in mammals.

[0326] 24. An anti-NOTCH4 antibody or a NOTCH4-binding fragment thereof that inhibits NOTCH4 activity.

[0327] 25. The antibody as described in Clause 24, wherein the antibody comprises a variety of polyclonal antibodies.

[0328] 26. The antibody as described in Clause 24, wherein the antibody is a monoclonal antibody or a NOTCH4-binding fragment thereof.

[0329] 27. The antibody according to any one of clauses 24 to 26, wherein the antibody comprises bovine, sheep, goat, camel, or human polyclonal or monoclonal antibodies, wherein at least a portion of the monoclonal antibody comprises an antibody sequence derived from a bovine, sheep, goat, or human antibody.

[0330] 28. The antibody described in Clause 26 includes bovine, sheep-like, goat-like, camel-like, or humanized antibodies or any antigen-binding fragment thereof.

[0331] 29. A pharmaceutical composition comprising an antibody according to any one of clauses 24 to 28 and a pharmaceutically acceptable carrier.

[0332] 30. The pharmaceutical composition described in Clause 29, for promoting milk production in mammals.

[0333] 31. A polynucleotide comprising an RNAi construct that inhibits the expression of ROBO2 or NOTCH4.

[0334] 32. The polynucleotide as described in Clause 31, comprising at least one non-naturally occurring nucleotide.

[0335] 33. The polynucleotide according to clause 31 or 32, comprising one or more of SEQ ID NO:32 to SEQ ID NO:35.

[0336] 34. A pharmaceutical composition comprising a polynucleotide according to any one of clauses 31 to 33.

[0337] 35. The pharmaceutical composition described in Clause 34, for promoting milk production in mammals.

[0338] 36. A transgenic mammal comprising gene modifications that produce one or more of the following phenotypes: expression of a soluble ROBO1 extracellular domain; inhibition of ROBO2 expression; and inhibition of NOTCH4 expression.

[0339] 37. The transgenic animal as described in Clause 36, wherein the phenotype is limited to mammary tissue.

[0340] 38. A genetically modified mammal as described in clause 36 or 37, wherein the genetically modified animal is a cow, sheep, goat, or camel.

[0341] 39. A transgenic mammal according to any one of clauses 36 to 38, comprising two gene modifications that produce two of the listed phenotypes.

[0342] 40. A transgenic mammal according to any one of clauses 36 to 38, comprising three gene modifications that produce all three of the listed phenotypes.

[0343] 41. A method for promoting milk production, the method comprising:

[0344] Administering a pharmaceutical composition that inhibits NOTCH4 activity to a transgenic mammal according to any one of claims 36 to 40.

[0345] 42. The method according to clause 41, wherein the pharmaceutical composition is the composition according to any one of clauses 22 to 23, 29 to 30 and 34 to 35.

[0346] 43. The method of claim 41, wherein the transgenic animal comprises a gene modification that expresses a soluble ROBO1 extracellular domain, the method further comprising administering to the transgenic animal a pharmaceutical composition according to any one of clauses 22 to 23, 29 to 30 and 34 to 35.

[0347] 44. The method of claim 41, wherein the transgenic mammal comprises a gene modification that inhibits the expression of ROBO2 and / or NOTCH4, the method further comprising administering to the transgenic animal the pharmaceutical composition according to clause 34 or 35. sequence list <110> University of California Board of Trustees Lindsay, Hinck S. Chatterjee, Sharmila Oscar Cazares Chen Min <120> Compositions and methods that can be used to promote milk production <130> UCSC-383PRV2 <160> 55 <170> PatentIn version 3.5 <210> 1 <211> 832 <212> PRT <213> Domestic cattle (Bos taurus) <400> 1 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Thr Ala Gly Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Val Gly Ser Glu Pro Pro His Phe Val Val Lys 305 310 315 320 Pro Arg Asp Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys 325 330 335 Glu Ala Thr Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly 340 345 350 Ser Gln Asn Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg 355 360 365 Phe Ser Val Ser Gln Thr Gly Asp Leu Thr Ile Thr Asn Val Gln Arg 370 375 380 Ser Asp Val Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser 385 390 395 400 Ile Ile Thr Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg 405 410 415 Pro Pro Pro Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val 420 425 430 Asp Gly Thr Leu Val Leu Ser Cys Val Ala Thr Gly Ser Pro Val Pro 435 440 445 Thr Ile Leu Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser 450 455 460 Arg Ile Lys Gln Leu Glu Thr Gly Val Leu Gln Ile Arg Tyr Ala Lys 465 470 475 480 Leu Gly Asp Thr Gly Arg Tyr Thr Cys Ile Ala Ser Thr Pro Ser Gly 485 490 495 Glu Ala Thr Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe Gly Val Pro 500 505 510 Val Gln Pro Pro Arg Pro Thr Asp Pro Asn Leu Ile Pro Ser Ala Pro 515 520 525 Ser Lys Pro Glu Val Thr Asp Val Ser Arg Asn Thr Val Thr Leu Ser 530 535 540 Trp Gln Pro Asn Leu Asn Ser Gly Ala Thr Pro Thr Ser Tyr Ile Ile 545 550 555 560 Glu Ala Phe Ser His Ala Ser Gly Ser Ser Trp Gln Thr Val Ala Glu 565 570 575 Asn Val Lys Thr Glu Thr Phe Ala Ile Lys Gly Leu Lys Pro Asn Ala 580 585 590 Ile Tyr Leu Phe Leu Val Arg Ala Ala Asn Ala Tyr Gly Ile Ser Asp 595 600 605 Pro Ser Gln Ile Ser Asp Pro Val Lys Thr Gln Asp Val Pro Pro Thr 610 615 620 Ser Gln Gly Val Asp His Lys Gln Val Gln Arg Glu Leu Gly Asn Val 625 630 635 640 Val Leu His Leu His Asn Pro Thr Ile Leu Ser Ser Ser Ser Ile Glu 645 650 655 Val His Trp Thr Val Asp Gln Gln Ser Gln Tyr Ile Gln Gly Tyr Lys 660 665 670 Val Leu Tyr Arg Pro Ser Gly Ala Asn His Gly Glu Ser Glu Trp Leu 675 680 685 Val Phe Glu Val Arg Thr Pro Thr Lys Asn Ser Val Val Ile Pro Asp 690 695 700 Leu Lys Lys Gly Val Asn Tyr Glu Ile Lys Ala Arg Pro Phe Phe Asn 705 710 715 720 Glu Phe Gln Gly Ala Asp Ser Glu Ile Lys Phe Ala Lys Thr Leu Glu 725 730 735 Glu Ala Pro Ser Ala Pro Pro Gln Ser Val Thr Val Ser Lys Asn Asp 740 745 750 Gly Asn Gly Thr Ala Ile Leu Val Ser Trp Gln Pro Pro Pro Glu Asp 755 760 765 Thr Gln Asn Gly Met Val Gln Glu Tyr Lys Val Trp Cys Leu Gly Asn 770 775 780 Glu Thr Arg Tyr His Ile Asn Lys Thr Val Asp Gly Ser Thr Phe Ser 785 790 795 800 Val Val Ile Pro Ser Leu Val Pro Gly Ile Arg Tyr Ser Val Glu Val 805 810 815 Ala Ala Ser Thr Gly Ala Gly Ser Gly Val Lys Ser Glu Pro Gln Phe 820 825 830 <210> 2 <211> 509 <212> PRT <213> Bos taurus <400> 2 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Thr Ala Gly Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Val Gly Ser Glu Pro Pro His Phe Val Val Lys 305 310 315 320 Pro Arg Asp Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys 325 330 335 Glu Ala Thr Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly 340 345 350 Ser Gln Asn Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg 355 360 365 Phe Ser Val Ser Gln Thr Gly Asp Leu Thr Ile Thr Asn Val Gln Arg 370 375 380 Ser Asp Val Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser 385 390 395 400 Ile Ile Thr Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg 405 410 415 Pro Pro Pro Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val 420 425 430 Asp Gly Thr Leu Val Leu Ser Cys Val Ala Thr Gly Ser Pro Val Pro 435 440 445 Thr Ile Leu Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser 450 455 460 Arg Ile Lys Gln Leu Glu Thr Gly Val Leu Gln Ile Arg Tyr Ala Lys 465 470 475 480 Leu Gly Asp Thr Gly Arg Tyr Thr Cys Ile Ala Ser Thr Pro Ser Gly 485 490 495 Glu Ala Thr Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe 500 505 <210> 3 <211> 225 <212> PRT <213> Bos taurus <400> 3 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe 225 <210> 4 <211> 832 <212> PRT <213> Homo sapiens <400> 4 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Thr Ala Gly Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Val Gly Ser Glu Pro Pro His Phe Val Val Lys 305 310 315 320 Pro Arg Asp Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys 325 330 335 Glu Ala Thr Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly 340 345 350 Ser Gln Asn Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg 355 360 365 Phe Ser Val Ser Gln Thr Gly Asp Leu Thr Ile Thr Asn Val Gln Arg 370 375 380 Ser Asp Val Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser 385 390 395 400 Ile Ile Thr Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg 405 410 415 Pro Pro Pro Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val 420 425 430 Asp Gly Thr Phe Val Leu Ser Cys Val Ala Thr Gly Ser Pro Val Pro 435 440 445 Thr Ile Leu Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser 450 455 460 Arg Ile Lys Gln Leu Glu Asn Gly Val Leu Gln Ile Arg Tyr Ala Lys 465 470 475 480 Leu Gly Asp Thr Gly Arg Tyr Thr Cys Ile Ala Ser Thr Pro Ser Gly 485 490 495 Glu Ala Thr Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe Gly Val Pro 500 505 510 Val Gln Pro Pro Arg Pro Thr Asp Pro Asn Leu Ile Pro Ser Ala Pro 515 520 525 Ser Lys Pro Glu Val Thr Asp Val Ser Arg Asn Thr Val Thr Leu Ser 530 535 540 Trp Gln Pro Asn Leu Asn Ser Gly Ala Thr Pro Thr Ser Tyr Ile Ile 545 550 555 560 Glu Ala Phe Ser His Ala Ser Gly Ser Ser Trp Gln Thr Val Ala Glu 565 570 575 Asn Val Lys Thr Glu Thr Ser Ala Ile Lys Gly Leu Lys Pro Asn Ala 580 585 590 Ile Tyr Leu Phe Leu Val Arg Ala Ala Asn Ala Tyr Gly Ile Ser Asp 595 600 605 Pro Ser Gln Ile Ser Asp Pro Val Lys Thr Gln Asp Val Leu Pro Thr 610 615 620 Ser Gln Gly Val Asp His Lys Gln Val Gln Arg Glu Leu Gly Asn Ala 625 630 635 640 Val Leu His Leu His Asn Pro Thr Val Leu Ser Ser Ser Ser Ile Glu 645 650 655 Val His Trp Thr Val Asp Gln Gln Ser Gln Tyr Ile Gln Gly Tyr Lys 660 665 670 Ile Leu Tyr Arg Pro Ser Gly Ala Asn His Gly Glu Ser Asp Trp Leu 675 680 685 Val Phe Glu Val Arg Thr Pro Ala Lys Asn Ser Val Val Ile Pro Asp 690 695 700 Leu Arg Lys Gly Val Asn Tyr Glu Ile Lys Ala Arg Pro Phe Phe Asn 705 710 715 720 Glu Phe Gln Gly Ala Asp Ser Glu Ile Lys Phe Ala Lys Thr Leu Glu 725 730 735 Glu Ala Pro Ser Ala Pro Pro Gln Gly Val Thr Val Ser Lys Asn Asp 740 745 750 Gly Asn Gly Thr Ala Ile Leu Val Ser Trp Gln Pro Pro Pro Glu Asp 755 760 765 Thr Gln Asn Gly Met Val Gln Glu Tyr Lys Val Trp Cys Leu Gly Asn 770 775 780 Glu Thr Arg Tyr His Ile Asn Lys Thr Val Asp Gly Ser Thr Phe Ser 785 790 795 800 Val Val Ile Pro Phe Leu Val Pro Gly Ile Arg Tyr Ser Val Glu Val 805 810 815 Ala Ala Ser Thr Gly Ala Gly Ser Gly Val Lys Ser Glu Pro Gln Phe 820 825 830 <210> 5 <211> 509 <212> PRT <213> Homo sapiens <400> 5 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Thr Ala Gly Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Val Gly Ser Glu Pro Pro His Phe Val Val Lys 305 310 315 320 Pro Arg Asp Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys 325 330 335 Glu Ala Thr Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly 340 345 350 Ser Gln Asn Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg 355 360 365 Phe Ser Val Ser Gln Thr Gly Asp Leu Thr Ile Thr Asn Val Gln Arg 370 375 380 Ser Asp Val Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser 385 390 395 400 Ile Ile Thr Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg 405 410 415 Pro Pro Pro Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val 420 425 430 Asp Gly Thr Phe Val Leu Ser Cys Val Ala Thr Gly Ser Pro Val Pro 435 440 445 Thr Ile Leu Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser [[ID= XVII]]450 455 460[[ID= XVIII]] [[ID= XIX]]Arg Ile Lys Gln Leu Glu Asn Gly Val Leu Gln Ile Arg Tyr Ala Lys[[ID= XX]] [[ID= XXI]]465 470 475 480[[ID= XXII]] [[ID= XXIII]]Leu Gly Asp Thr Gly Arg Tyr Thr Cys Ile Ala Ser Thr Pro Ser Gly[[ID= XXIV]] [[ID= XXV]]485 490 49五十[[ID= XXVI]] [[ID= XXVII]]Glu Ala Thr Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe[[ID= XXVIII]] [[ID= XXIX]]500 505[[ID= XXX]] [[ID= XXXI]]<210> 6[[ID= XXXII]] [[ID= XXXIII]]<211> 225[[ID= XXXIV]] [[ID= XXXV]]<212> PRT[[ID= XXXVI]] [[ID= XXXVII]]<213> Homo sapiens[[ID= XXXVIII]] [[ID= XXXIX]]<400> 6[[ID= XL]] [[ID= XLI]]Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu[[ID= XLII]] [[ID= XLIII]]1 5 10 15[[ID= XLIV]] It should be noted that there seems to be an error in the original text where "49五十" is likely a typo and should be "495". The above translation is based on the corrected understanding.Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe 225 <210> 7 <211> 829 <​​​​​​​​​​​​​​​​​​​​​​​​​Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Ile Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Thr Ala Gly Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Glu Pro Pro His Phe Val Val Lys Pro Arg Asp 305 310 315 320 Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys Glu Ala Thr 325 330 335 Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly Ser Gln Asn 340 345 350 Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg Phe Ser Val 355 360 365 Ser Gln Thr Gly Asp Leu Thr Ile Thr Asn Val Gln Arg Ser Asp Val 370 375 380 Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser Ile Ile Thr 385 390 395 400 Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg Pro Pro Pro 405 410 415 Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val Asp Gly Thr 420 425 430 Leu Ile Leu Ser Cys Val Ala Thr Gly Ser Pro Ala Pro Thr Ile Leu 435 440 445 Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser Arg Ile Lys 450 455 460 Gln Leu Glu Ser Gly Val Leu Gln Ile Arg Tyr Ala Lys Leu Gly Asp 465 470 475 480 Thr Gly Arg Tyr Thr Cys Thr Ala Ser Thr Pro Ser Gly Glu Ala Thr 485 490 495 Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe Gly Val Pro Val Gln Pro 500 505 510 Pro Arg Pro Thr Asp Pro Asn Leu Ile Pro Ser Ala Pro Ser Lys Pro 515 520 525 Glu Val Thr Asp Val Ser Lys Asn Thr Val Thr Leu Ser Trp Gln Pro 530 535 540 Asn Leu Asn Ser Gly Ala Thr Pro Thr Ser Tyr Ile Ile Glu Ala Phe 545 550 555 560 Ser His Ala Ser Gly Ser Ser Trp Gln Thr Ala Ala Glu Asn Val Lys 565 570 575 Thr Glu Thr Phe Ala Ile Lys Gly Leu Lys Pro Asn Ala Ile Tyr Leu 580 585 590 Phe Leu Val Arg Ala Ala Asn Ala Tyr Gly Ile Ser Asp Pro Ser Gln 595 600 605 Ile Ser Asp Pro Val Lys Thr Gln Asp Val Pro Pro Thr Ser Gln Gly 610 615 620 Val Asp His Lys Gln Val Gln Arg Glu Leu Gly Asn Val Val Leu His 625 630 635 640 Leu His Asn Pro Thr Ile Leu Ser Ser Ser Ser Val Glu Val His Trp 645 650 655 Thr Val Asp Gln Gln Ser Gln Tyr Ile Gln Gly Tyr Lys Ile Leu Tyr 660 665 670 Arg Pro Ser Gly Ala Ser His Gly Glu Ser Glu Trp Leu Val Phe Glu 675 680 685 Val Arg Thr Pro Thr Lys Asn Ser Val Val Ile Pro Asp Leu Arg Lys 690 695 700 Gly Val Asn Tyr Glu Ile Lys Ala Arg Pro Phe Phe Asn Glu Phe Gln 705 710 715 720 Gly Ala Asp Ser Glu Ile Lys Phe Ala Lys Thr Leu Glu Glu Ala Pro 725 730 735 Ser Ala Pro Pro Arg Ser Val Thr Val Ser Lys Asn Asp Gly Asn Gly 740 745 750 Thr Ala Ile Leu Val Thr Trp Gln Pro Pro Pro Glu Asp Thr Gln Asn 755 760 765 Gly Met Val Gln Glu Tyr Lys Val Trp Cys Leu Gly Asn Glu Thr Lys 770 775 780 Tyr His Ile Asn Lys Thr Val Asp Gly Ser Thr Phe Ser Val Val Ile 785 790 795 800 Pro Ser Leu Val Pro Gly Ile Arg Tyr Ser Val Glu Val Ala Ala Ser 805 810 815 Thr Gly Ala Gly Pro Gly Val Lys Ser Glu Pro Gln Phe 820 825 <210> 8 <211> 506 <212> PRT <213> Bison bison <400> 8 Met Ile Ala Glu Pro Ala Arg Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Ile Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Thr Ala Gly Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Glu Pro Pro His Phe Val Val Lys Pro Arg Asp 305 310 315 320 Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys Glu Ala Thr 325 330 335 Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly Ser Gln Asn 340 345 350 Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg Phe Ser Val 355 360 365 Ser Gln Thr Gly Asp Leu Thr Ile Thr Asn Val Gln Arg Ser Asp Val 370 375 380 Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser Ile Ile Thr 385 390 395 400 Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg Pro Pro Pro 405 410 415 Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val Asp Gly Thr 420 425 430 Leu Ile Leu Ser Cys Val Ala Thr Gly Ser Pro Ala Pro Thr Ile Leu 435 440 445 Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser Arg Ile Lys 450 455 460 Gln Leu Glu Ser Gly Val Leu Gln Ile Arg Tyr Ala Lys Leu Gly Asp 465 470 475 480 Thr Gly Arg Tyr Thr Cys Thr Ala Ser Thr Pro Ser Gly Glu Ala Thr 485 490 495 Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe 500 505 <210> 9 <211> 225 <212> PRT <213> Bison bison <400> 9 Met Ile Ala Glu Pro Ala Arg Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Ile Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe 225 <210> 10 <211> 832 <212> PRT <213> Bactrian camel (Camelus bactrianus) <400> 10 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Ser Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Met Ala Ser Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Val Gly Ser Glu Pro Pro His Phe Val Val Lys 305 310 315 320 Pro Arg Asp Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys 325 330 335 Glu Ala Thr Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly 340 345 350 Ser Gln Asn Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg 355 360 365 Phe Ser Val Ser Gln Thr Gly Asp Leu Thr Ile Thr Asn Val Gln Arg 370 375 380 Ser Asp Val Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser 385 390 395 400 Ile Ile Thr Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg 405 410 415 Pro Pro Pro Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val 420 425 430 Asp Gly Thr Leu Val Leu Ser Cys Val Ala Thr Gly Ser Pro Val Pro 435 440 445 Thr Ile Leu Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser 450 455 460 Arg Ile Lys Gln Leu Glu Thr Gly Val Leu Gln Ile Arg Tyr Ala Lys 465 470 475 480 Leu Gly Asp Thr Gly Arg Tyr Thr Cys Ile Ala Ser Thr Pro Ser Gly 485 490 495 Glu Ala Thr Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe Gly Val Pro 500 505 510 Val Gln Pro Pro Arg Pro Thr Asp Pro Asn Leu Ile Pro Ser Ala Pro 515 520 525 Ser Lys Pro Glu Val Thr Asp Val Ser Arg Asn Thr Val Thr Leu Ser 530 535 540 Trp Gln Pro Asn Leu Asn Ser Gly Ala Thr Pro Thr Ser Tyr Ile Ile 545 550 555 560 Glu Ala Phe Ser His Ala Ser Gly Ser Ser Trp Gln Thr Val Ala Glu 565 570 575 Asn Val Lys Met Glu Thr Phe Ala Val Lys Gly Leu Lys Pro Asn Ala 580 585 590 Ile Tyr Leu Phe Leu Val Arg Ala Ala Asn Ala Tyr Gly Ile Ser Asp 595 600 605 Pro Ser Gln Ile Ser Asp Pro Val Lys Thr Gln Asp Val Pro Pro Thr 610 615 620 Ser Gln Gly Val Asp His Lys Gln Val Gln Arg Glu Leu Gly Asn Val 625 630 635 640 Val Leu His Leu His Asn Pro Thr Ile Leu Ser Ser Ser Ser Ile Glu 645 650 655 Val His Trp Thr Val Asp Gln Gln Ser Gln Tyr Ile Gln Gly Tyr Lys 660 665 670 Ile Leu Tyr Arg Pro Ser Gly Ala Asn His Gly Glu Ser Gly Trp Leu 675 680 685 Val Phe Glu Val Arg Thr Pro Thr Lys Asn Ser Val Val Ile Pro Asp 690 695 700 Leu Lys Lys Gly Val Asn Tyr Glu Ile Lys Ala Arg Pro Phe Phe Asn 705 710 715 720 Glu Phe Gln Gly Ala Asp Ser Glu Ile Lys Phe Ala Lys Thr Leu Glu 725 730 735 Glu Ala Pro Ser Ala Pro Pro Gln Ser Val Thr Val Ser Lys Asn Asp 740 745 750 Gly Asn Gly Thr Ala Ile Leu Val Ser Trp Gln Pro Pro Pro Glu Asp 755 760 765 Thr Gln Asn Gly Met Val Gln Glu Tyr Lys Val Trp Cys Leu Gly Asn 770 775 780 Glu Thr Arg Tyr His Ile Asn Lys Thr Val Asp Gly Ser Thr Phe Ser 785 790 795 800 Val Val Ile Pro Ser Leu Val Pro Gly Ile Arg Tyr Ser Val Glu Val 805 810 815 Ala Ala Ser Thr Gly Ala Gly Ser Gly Val Lys Ser Glu Pro Gln Phe 820 825 830 <210> 11 <211> 509 <212> PRT <213> Bactrian Camel (Camelus bactrianus) <400> 11 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Ser Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Met Ala Ser Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Val Gly Ser Glu Pro Pro His Phe Val Val Lys 305 310 315 320 Pro Arg Asp Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys 325 330 335 Glu Ala Thr Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly 340 345 350 Ser Gln Asn Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg 355 360 365 Phe Ser Val Ser Gln Thr Gly Asp Leu Thr Ile Thr Asn Val Gln Arg 370 375 380 Ser Asp Val Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser 385 390 395 400 Ile Ile Thr Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg 405 410 415 Pro Pro Pro Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val 420 425 430 Asp Gly Thr Leu Val Leu Ser Cys Val Ala Thr Gly Ser Pro Val Pro 435 440 445 Thr Ile Leu Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser 450 455 460 Arg Ile Lys Gln Leu Glu Thr Gly Val Leu Gln Ile Arg Tyr Ala Lys 465 470 475 480 Leu Gly Asp Thr Gly Arg Tyr Thr Cys Ile Ala Ser Thr Pro Ser Gly 485 490 495 Glu Ala Thr Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe 500 505 <210> 12 <211> 225 <212> PRT <213> Bactrian camel (Camelus bactrianus) <400> 12 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Ser Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe 225 <210> 13 <211> 832 <212> PRT <213> Goat (Capra hircus) <400> 13 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Phe Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Thr Ala Gly Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Val Gly Ser Glu Pro Pro His Phe Val Val Lys 305 310 315 320 Pro Arg Asp Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys 325 330 335 Glu Ala Thr Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly 340 345 350 Ser Gln Asn Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg 355 360 365 Phe Ser Val Ser Gln Thr Gly Asp Leu Thr Ile Thr Asn Val Gln Arg 370 375 380 Ser Asp Val Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser 385 390 395 400 Ile Ile Thr Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg 405 410 415 Pro Pro Pro Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val 420 425 430 Asp Gly Thr Leu Val Leu Ser Cys Val Ala Thr Gly Ser Pro Val Pro 435 440 445 Thr Ile Leu Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser 450 455 460 Arg Ile Lys Gln Leu Glu Thr Gly Val Leu Gln Ile Arg Tyr Ala Lys 465 470 475 480 Leu Gly Asp Thr Gly Arg Tyr Thr Cys Ile Ala Ser Thr Pro Ser Gly 485 490 495 Glu Ala Thr Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe Gly Val Pro 500 505 510 Val Gln Pro Pro Arg Pro Thr Asp Pro Asn Leu Ile Pro Ser Ala Pro 515 520 525 Ser Lys Pro Glu Val Thr Asp Val Ser Arg Asn Thr Val Thr Leu Ser 530 535 540 Trp Gln Pro Asn Leu Asn Ser Gly Ala Thr Pro Thr Ser Tyr Ile Ile 545 550 555 560 Glu Ala Phe Ser His Ala Ser Gly Ser Ser Trp Gln Thr Val Ala Glu 565 570 575 Asn Val Lys Thr Glu Thr Phe Ala Ile Lys Gly Leu Lys Pro Asn Ala 580 585 590 Ile Tyr Leu Phe Leu Val Arg Ala Ala Asn Ala Tyr Gly Ile Ser Asp 595 600 605 Pro Ser Gln Ile Ser Asp Pro Val Lys Thr Gln Asp Ile Pro Pro Thr 610 615 620 Ser Gln Gly Val Asp His Lys Gln Val Gln Arg Glu Leu Gly Asn Val 625 630 635 640 Val Leu His Leu His Asn Pro Thr Ile Leu Ser Ser Ser Ser Ile Glu 645 650 655 Val His Trp Thr Val Asp Gln Gln Ser Gln Tyr Ile Gln Gly Tyr Lys 660 665 670 Val Leu Tyr Arg Pro Ser Gly Ala Asn His Gly Glu Ser Glu Trp Leu 675 680 685 Val Phe Glu Val Arg Thr Pro Thr Lys Asn Ser Val Val Ile Pro Asp 690 695 700 Leu Lys Lys Gly Val Asn Tyr Glu Ile Lys Ala Arg Pro Phe Phe Asn 705 710 715 720 Glu Phe Gln Gly Ala Asp Ser Glu Ile Lys Phe Ala Lys Thr Leu Glu 725 730 735 Glu Ala Pro Ser Ala Pro Pro Gln Ser Val Thr Val Ser Lys Asn Asp 740 745 750 Gly Asn Gly Thr Ala Ile Leu Val Ser Trp Gln Pro Pro Pro Glu Asp 755 760 765 Thr Gln Asn Gly Met Val Gln Glu Tyr Lys Val Trp Cys Leu Gly Asn 770 775 780 Glu Thr Arg Tyr His Ile Asn Lys Thr Val Asp Gly Ser Thr Phe Ser 785 790 795 800 Val Val Ile Pro Ser Leu Val Pro Gly Ile Arg Tyr Ser Val Glu Val 805 810 815 Ala Ala Ser Thr Gly Ala Gly Ser Gly Val Lys Ser Glu Pro Gln Phe 820 825 830 <210> 14 <211> 509 <212> PRT <213> Goat (Capra hircus) <400> 14 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Phe Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Thr Ala Gly Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Val Gly Ser Glu Pro Pro His Phe Val Val Lys 305 310 315 320 Pro Arg Asp Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys 325 330 335 Glu Ala Thr Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly 340 345 350 Ser Gln Asn Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg 355 360 365 Phe Ser Val Ser Gln Thr Gly Asp Leu Thr Ile Thr Asn Val Gln Arg 370 375 380 Ser Asp Val Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser 385 390 395 400 Ile Ile Thr Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg 405 410 415 Pro Pro Pro Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val 420 425 430 Asp Gly Thr Leu Val Leu Ser Cys Val Ala Thr Gly Ser Pro Val Pro 435 440 445 Thr Ile Leu Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser 450 455 460 Arg Ile Lys Gln Leu Glu Thr Gly Val Leu Gln Ile Arg Tyr Ala Lys 465 470 475 480 Leu Gly Asp Thr Gly Arg Tyr Thr Cys Ile Ala Ser Thr Pro Ser Gly 485 490 495 Glu Ala Thr Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe 500 505 <210> 15 <211> 225 <212> PRT <213> Goat (Capra hircus) <400> 15 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Phe Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe 225 <210> 16 <211> 829 <212> PRT <213> Sheep (Ovis aries) <400> 16 Met Ile Ala Glu Pro Ala Arg Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Ile Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Thr Ala Gly Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Glu Pro Pro His Phe Val Val Lys Pro Arg Asp 305 310 315 320 Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys Glu Ala Thr 325 330 335 Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly Ser Gln Asn 340 345 350 Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg Phe Ser Val 355 360 365 Ser Gln Thr Gly Asp Leu Thr Ile Thr Asn Val Gln Arg Ser Asp Val 370 375 380 Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser Ile Ile Thr 385 390 395 400 Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg Pro Pro Pro 405 410 415 Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val Asp Gly Thr 420 425 430 Leu Ile Leu Ser Cys Val Ala Thr Gly Ser Pro Ala Pro Thr Ile Leu 435 440 445 Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser Arg Ile Lys 450 455 460 Gln Leu Glu Ser Gly Val Leu Gln Ile Arg Tyr Ala Lys Leu Gly Asp 465 470 475 480 Thr Gly Arg Tyr Thr Cys Thr Ala Ser Thr Pro Ser Gly Glu Ala Thr 485 490 495 Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe Gly Val Pro Val Gln Pro 500 505 510 Pro Arg Pro Thr Asp Pro Asn Leu Ile Pro Ser Ala Pro Ser Lys Pro 515 520 525 Glu Val Thr Asp Val Ser Lys Asn Thr Val Thr Leu Ser Trp Gln Pro 530 535 540 Asn Leu Asn Ser Gly Ala Thr Pro Thr Ser Tyr Ile Ile Glu Ala Phe 545 550 555 560 Ser His Ala Ser Gly Ser Ser Trp Gln Thr Ala Ala Glu Asn Val Lys 565 570 575 Thr Glu Thr Phe Ala Ile Lys Gly Leu Lys Pro Asn Ala Ile Tyr Leu 580 585 590 Phe Leu Val Arg Ala Ala Asn Ala Tyr Gly Ile Ser Asp Pro Ser Gln 595 600 605 Ile Ser Asp Pro Val Lys Thr Gln Asp Val Pro Pro Thr Ser Gln Gly 610 615 620 Val Asp His Lys Gln Val Gln Arg Glu Leu Gly Asn Val Val Leu His 625 630 635 640 Leu His Asn Pro Thr Ile Leu Ser Ser Ser Ser Val Glu Val His Trp 645 650 655 Thr Val Asp Gln Gln Ser Gln Tyr Ile Gln Gly Tyr Lys Ile Leu Tyr 660 665 670 Arg Pro Ser Gly Ala Ser His Gly Glu Ser Glu Trp Leu Val Phe Glu 675 680 685 Val Arg Thr Pro Thr Lys Asn Ser Val Val Ile Pro Asp Leu Arg Lys 690 695 700 Gly Val Asn Tyr Glu Ile Lys Ala Arg Pro Phe Phe Asn Glu Phe Gln 705 710 715 720 Gly Ala Asp Ser Glu Ile Lys Phe Ala Lys Thr Leu Glu Glu Ala Pro 725 730 735 Ser Ala Pro Pro Arg Ser Val Thr Val Ser Lys Asn Asp Gly Asn Gly 740 745 750 Thr Ala Ile Leu Val Thr Trp Gln Pro Pro Pro Glu Asp Thr Gln Asn 755 760 765 Gly Met Val Gln Glu Tyr Lys Val Trp Cys Leu Gly Asn Glu Thr Lys 770 775 780 Tyr His Ile Asn Lys Thr Val Asp Gly Ser Thr Phe Ser Val Val Ile 785 790 795 800 Pro Ser Leu Val Pro Gly Ile Arg Tyr Ser Val Glu Val Ala Ala Ser 805 810 815 Thr Gly Ala Gly Pro Gly Val Lys Ser Glu Pro Gln Phe 820 825 <210> 17 <211> 506 <212> PRT <213> Sheep (Ovis aries) <400> 17 Met Ile Ala Glu Pro Ala Arg Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Ile Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Thr Ala Gly Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Glu Pro Pro His Phe Val Val Lys Pro Arg Asp 305 310 315 320 Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys Glu Ala Thr 325 330 335 Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly Ser Gln Asn 340 345 350 Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg Phe Ser Val 355 360 365 Ser Gln Thr Gly Asp Leu Thr Ile Thr Asn Val Gln Arg Ser Asp Val 370 375 380 Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser Ile Ile Thr 385 390 395 400 Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg Pro Pro Pro 405 410 415 Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val Asp Gly Thr 420 425 430 Leu Ile Leu Ser Cys Val Ala Thr Gly Ser Pro Ala Pro Thr Ile Leu 435 440 445 Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser Arg Ile Lys 450 455 460 Gln Leu Glu Ser Gly Val Leu Gln Ile Arg Tyr Ala Lys Leu Gly Asp 465 470 475 480 Thr Gly Arg Tyr Thr Cys Thr Ala Ser Thr Pro Ser Gly Glu Ala Thr 485 490 495 Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe 500 505 <210> 18 <211> 225 <212> PRT <213> Sheep (Ovis aries) <400> 18 Met Ile Ala Glu Pro Ala Arg Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Ile Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe 225 <210> 19 <211> 832 <212> PRT <213> Wild yak (Bos Mutas) <400> 19 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Thr Ala Gly Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Val Gly Ser Glu Pro Pro His Phe Val Val Lys 305 310 315 320 Pro Arg Asp Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys 325 330 335 Glu Ala Thr Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly 340 345 350 Ser Gln Asn Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg 355 360 365 Phe Ser Val Ser Gln Thr Gly Asp Leu Thr Ile Thr Asn Val Gln Arg 370 375 380 Ser Asp Val Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser 385 390 395 400 Ile Ile Thr Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg 405 410 415 Pro Pro Pro Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val 420 425 430 Asp Gly Thr Leu Val Leu Ser Cys Val Ala Thr Gly Ser Pro Val Pro 435 440 445 Thr Ile Leu Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser 450 455 460 Arg Ile Lys Gln Leu Glu Thr Gly Val Leu Gln Ile Arg Tyr Ala Lys 465 470 475 480 Leu Gly Asp Thr Gly Arg Tyr Thr Cys Ile Ala Ser Thr Pro Ser Gly 485 490 495 Glu Ala Thr Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe Gly Val Pro 500 505 510 Val Gln Pro Pro Arg Pro Thr Asp Pro Asn Leu Ile Pro Ser Ala Pro 515 520 525 Ser Lys Pro Glu Val Thr Asp Val Ser Arg Asn Thr Val Thr Leu Ser 530 535 540 Trp Gln Pro Asn Leu Asn Ser Gly Ala Thr Pro Thr Ser Tyr Ile Ile 545 550 555 560 Glu Ala Phe Ser His Ala Ser Gly Ser Ser Trp Gln Thr Val Ala Glu 565 570 575 Asn Val Lys Thr Glu Thr Phe Ala Ile Lys Gly Leu Lys Pro Asn Ala 580 585 590 Ile Tyr Leu Phe Leu Val Arg Ala Ala Asn Ala Tyr Gly Ile Ser Asp 595 600 605 Pro Ser Gln Ile Ser Asp Pro Val Lys Thr Gln Asp Val Pro Pro Thr 610 615 620 Ser Gln Gly Val Asp His Lys Gln Val Gln Arg Glu Leu Gly Asn Val 625 630 635 640 Val Leu His Leu His Asn Pro Thr Ile Leu Ser Ser Ser Ser Ile Glu 645 650 655 Val His Trp Thr Val Asp Gln Gln Ser Gln Tyr Ile Gln Gly Tyr Lys 660 665 670 Val Leu Tyr Arg Pro Ser Gly Ala Asn His Gly Glu Ser Glu Trp Leu 675 680 685 Val Phe Glu Val Arg Thr Pro Thr Lys Asn Ser Val Val Ile Pro Asp 690 695 700 Leu Lys Lys Gly Val Asn Tyr Glu Ile Lys Ala Arg Pro Phe Phe Asn 705 710 715 720 Glu Phe Gln Gly Ala Asp Ser Glu Ile Lys Phe Ala Lys Thr Leu Glu 725 730 735 Glu Ala Pro Ser Ala Pro Pro Gln Ser Val Thr Val Ser Lys Asn Asp 740 745 750 Gly Asn Gly Thr Ala Ile Leu Val Ser Trp Gln Pro Pro Pro Glu Asp 755 760 765 Thr Gln Asn Gly Met Val Gln Glu Tyr Lys Val Trp Cys Leu Gly Asn 770 775 780 Glu Thr Arg Tyr His Ile Asn Lys Thr Val Asp Gly Ser Thr Phe Ser 785 790 795 800 Val Val Ile Pro Ser Leu Val Pro Gly Ile Arg Tyr Ser Val Glu Val 805 810 815 Ala Ala Ser Thr Gly Ala Gly Ser Gly Val Lys Ser Glu Pro Gln Phe 820 825 830 <210> 20 <211> 509 <212> PRT <213> Bos Mutas <400> 20 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn ​​​​​ Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Thr Ala Gly Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Val Gly Ser Glu Pro Pro His Phe Val Val Lys 305 310 315 320 Pro Arg Asp Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys 325 330 335 Glu Ala Thr Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly 340 345 350 Ser Gln Asn Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg 355 360 365 Phe Ser Val Ser Gln Thr Gly Asp Leu Thr Ile Thr Asn Val Gln Arg 370 375 380 Ser Asp Val Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser 385 390 395 400 Ile Ile Thr Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg 405 410 415 Pro Pro Pro Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val 420 425 430 Asp Gly Thr Leu Val Leu Ser Cys Val Ala Thr Gly Ser Pro Val Pro 435 440 445 Thr Ile Leu Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser 450 455 460 Arg Ile Lys Gln Leu Glu Thr Gly Val Leu Gln Ile Arg Tyr Ala Lys 465 470 475 480 Leu Gly Asp Thr Gly Arg Tyr Thr Cys Ile Ala Ser Thr Pro Ser Gly 485 490 495 Glu Ala Thr Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe 500 505 <210> 21 <211> 225 <212> PRT <213> Bos Mutas <400> 21 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe 225 <210> 22 <211> 829 <212> PRT <213> House mouse (Mus musculus) <400> 22 Met Ile Ala Glu Pro Ala Arg Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Ile Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Thr Ala Gly Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Glu Pro Pro His Phe Val Val Lys Pro Arg Asp 305 310 315 320 Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys Glu Ala Thr 325 330 335 Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly Ser Gln Asn 340 345 350 Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg Phe Ser Val 355 360 365 Ser Gln Thr Gly Asp Leu Thr Ile Thr Asn Val Gln Arg Ser Asp Val 370 375 380 Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser Ile Ile Thr 385 390 395 400 Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg Pro Pro Pro 405 410 415 Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val Asp Gly Thr 420 425 430 Leu Ile Leu Ser Cys Val Ala Thr Gly Ser Pro Ala Pro Thr Ile Leu 435 440 445 Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser Arg Ile Lys 450 455 460 Gln Leu Glu Ser Gly Val Leu Gln Ile Arg Tyr Ala Lys Leu Gly Asp 465 470 475 480 Thr Gly Arg Tyr Thr Cys Thr Ala Ser Thr Pro Ser Gly Glu Ala Thr 485 490 495 Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe Gly Val Pro Val Gln Pro 500 505 510 Pro Arg Pro Thr Asp Pro Asn Leu Ile Pro Ser Ala Pro Ser Lys Pro 515 520 525 Glu Val Thr Asp Val Ser Lys Asn Thr Val Thr Leu Ser Trp Gln Pro 530 535 540 Asn Leu Asn Ser Gly Ala Thr Pro Thr Ser Tyr Ile Ile Glu Ala Phe 545 550 555 560 Ser His Ala Ser Gly Ser Ser Trp Gln Thr Ala Ala Glu Asn Val Lys 565 570 575 Thr Glu Thr Phe Ala Ile Lys Gly Leu Lys Pro Asn Ala Ile Tyr Leu 580 585 590 Phe Leu Val Arg Ala Ala Asn Ala Tyr Gly Ile Ser Asp Pro Ser Gln 595 600 605 Ile Ser Asp Pro Val Lys Thr Gln Asp Val Pro Pro Thr Ser Gln Gly 610 615 620 Val Asp His Lys Gln Val Gln Arg Glu Leu Gly Asn Val Val Leu His 625 630 635 640 Leu His Asn Pro Thr Ile Leu Ser Ser Ser Ser Val Glu Val His Trp 645 650 655 Thr Val Asp Gln Gln Ser Gln Tyr Ile Gln Gly Tyr Lys Ile Leu Tyr 660 665 670 Arg Pro Ser Gly Ala Ser His Gly Glu Ser Glu Trp Leu Val Phe Glu 675 680 685 Val Arg Thr Pro Thr Lys Asn Ser Val Val Ile Pro Asp Leu Arg Lys 690 695 700 Gly Val Asn Tyr Glu Ile Lys Ala Arg Pro Phe Phe Asn Glu Phe Gln 705 710 715 720 Gly Ala Asp Ser Glu Ile Lys Phe Ala Lys Thr Leu Glu Glu Ala Pro 725 730 735 Ser Ala Pro Pro Arg Ser Val Thr Val Ser Lys Asn Asp Gly Asn Gly 740 745 750 Thr Ala Ile Leu Val Thr Trp Gln Pro Pro Pro Glu Asp Thr Gln Asn 755 760 765 Gly Met Val Gln Glu Tyr Lys Val Trp Cys Leu Gly Asn Glu Thr Lys 770 775 780 Tyr His Ile Asn Lys Thr Val Asp Gly Ser Thr Phe Ser Val Val Ile 785 790 795 800 Pro Ser Leu Val Pro Gly Ile Arg Tyr Ser Val Glu Val Ala Ala Ser 805 810 815 Thr Gly Ala Gly Pro Gly Val Lys Ser Glu Pro Gln Phe 820 825 <210> 23 <211> 506 <212> PRT <213> Mus musculus <400> 23 Met Ile Ala Glu Pro Ala Arg Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Ile Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Thr Ala Gly Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Glu Pro Pro His Phe Val Val Lys Pro Arg Asp 305 310 315 320 Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys Glu Ala Thr 325 330 335 Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly Ser Gln Asn 340 345 350 Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg Phe Ser Val 355 360 365 Ser Gln Thr Gly Asp Leu Thr Ile Thr Asn Val Gln Arg Ser Asp Val 370 375 380 Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser Ile Ile Thr 385 390 395 400 Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg Pro Pro Pro 405 410 415 Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val Asp Gly Thr 420 425 430 Leu Ile Leu Ser Cys Val Ala Thr Gly Ser Pro Ala Pro Thr Ile Leu 435 440 445 Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser Arg Ile Lys 450 455 460 Gln Leu Glu Ser Gly Val Leu Gln Ile Arg Tyr Ala Lys Leu Gly Asp 465 470 475 480 Thr Gly Arg Tyr Thr Cys Thr Ala Ser Thr Pro Ser Gly Glu Ala Thr 485 490 495 Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe 500 505 <210> 24 <211> 225 <212> PRT <213> Mus musculus <400> 24 Met Ile Ala Glu Pro Ala Arg Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Ile Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe 225 <210> 25 <211> 829 <212> PRT <213> Rattus norvegicus <400> 25 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Ile Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Thr Ala Gly Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Glu Pro Pro His Phe Val Val Lys Pro Arg Asp 305 310 315 320 Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys Glu Ala Thr 325 330 335 Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly Ser Gln Asn 340 345 350 Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg Phe Ser Val 355 360 365 Ser Gln Thr Gly Asp Leu Thr Val Thr Asn Val Gln Arg Ser Asp Val 370 375 380 Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser Ile Ile Thr 385 390 395 400 Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg Pro Pro Pro 405 410 415 Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val Asp Gly Thr 420 425 430 Leu Thr Leu Ser Cys Val Ala Thr Gly Ser Pro Val Pro Thr Ile Leu 435 440 445 Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser Arg Ile Lys 450 455 460 Gln Leu Glu Ser Gly Val Leu Gln Ile Arg Tyr Ala Lys Leu Gly Asp 465 470 475 480 Thr Gly Arg Tyr Thr Cys Thr Ala Ser Thr Pro Ser Gly Glu Ala Thr 485 490 495 Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe Gly Val Pro Val Gln Pro 500 505 510 Pro Arg Pro Thr Asp Pro Asn Leu Ile Pro Ser Ala Pro Ser Lys Pro 515 520 525 Glu Val Thr Asp Val Ser Lys Asn Thr Val Thr Leu Leu Trp Gln Pro 530 535 540 Asn Leu Asn Ser Gly Ala Thr Pro Thr Ser Tyr Ile Ile Glu Ala Phe 545 550 555 560 Ser His Ala Ser Gly Ser Ser Trp Gln Thr Val Ala Glu Asn Val Lys 565 570 575 Thr Glu Thr Phe Ala Ile Lys Gly Leu Lys Pro Asn Ala Ile Tyr Leu 580 585 590 Phe Leu Val Arg Ala Ala Asn Ala Tyr Gly Ile Ser Asp Pro Ser Gln 595 600 605 Ile Ser Asp Pro Val Lys Thr Gln Asp Val Pro Pro Thr Thr Gln Gly 610 615 620 Val Asp His Lys Gln Val Gln Arg Glu Leu Gly Asn Val Val Leu His 625 630 635 640 Leu His Asn Pro Thr Ile Leu Ser Ser Ser Ser Val Glu Val His Trp 645 650 655 Thr Val Asp Gln Gln Ser Gln Tyr Ile Gln Gly Tyr Lys Ile Leu Tyr 660 665 670 Arg Pro Ser Gly Ala Ser His Gly Glu Ser Glu Trp Leu Val Phe Glu 675 680 685 Val Arg Thr Pro Thr Lys Asn Ser Val Val Ile Pro Asp Leu Arg Lys 690 695 700 Gly Val Asn Tyr Glu Ile Lys Ala Arg Pro Phe Phe Asn Glu Phe Gln 705 710 715 720 Gly Ala Asp Ser Glu Ile Lys Phe Ala Lys Thr Leu Glu Glu Ala Pro 725 730 735 Ser Ala Pro Pro Arg Ser Val Thr Val Ser Lys Asn Asp Gly Asn Gly 740 745 750 Thr Ala Ile Leu Val Thr Trp Gln Pro Pro Pro Glu Asp Thr Gln Asn 755 760 765 Gly Met Val Gln Glu Tyr Lys Val Trp Cys Leu Gly Asn Glu Thr Arg 770 775 780 Tyr His Ile Asn Lys Thr Val Asp Gly Ser Thr Phe Ser Val Val Ile 785 790 795 800 Pro Ser Leu Val Pro Gly Ile Arg Tyr Ser Val Glu Val Ala Ala Ser 805 810 815 Thr Gly Ala Gly Pro Gly Val Lys Ser Glu Pro Gln Phe 820 825 <210> 26 <211> 506 <212> PRT <213> Rattus norvegicus <400> 26 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Ile Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe Val Lys Arg Pro Ser Asn Leu Ala Val Thr Val Asp Asp Ser Ala 225 230 235 240 Glu Phe Lys Cys Glu Ala Arg Gly Asp Pro Val Pro Thr Val Arg Trp 245 250 255 Arg Lys Asp Asp Gly Glu Leu Pro Lys Ser Arg Tyr Glu Ile Arg Asp 260 265 270 Asp His Thr Leu Lys Ile Arg Lys Val Thr Ala Gly Asp Met Gly Ser 275 280 285 Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys Ala Glu Ala Ser Ala 290 295 300 Thr Leu Thr Val Gln Glu Pro Pro His Phe Val Val Lys Pro Arg Asp 305 310 315 320 Gln Val Val Ala Leu Gly Arg Thr Val Thr Phe Gln Cys Glu Ala Thr 325 330 335 Gly Asn Pro Gln Pro Ala Ile Phe Trp Arg Arg Glu Gly Ser Gln Asn 340 345 350 Leu Leu Phe Ser Tyr Gln Pro Pro Gln Ser Ser Ser Arg Phe Ser Val 355 360 365 Ser Gln Thr Gly Asp Leu Thr Val Thr Asn Val Gln Arg Ser Asp Val 370 375 380 Gly Tyr Tyr Ile Cys Gln Thr Leu Asn Val Ala Gly Ser Ile Ile Thr 385 390 395 400 Lys Ala Tyr Leu Glu Val Thr Asp Val Ile Ala Asp Arg Pro Pro Pro 405 410 415 Val Ile Arg Gln Gly Pro Val Asn Gln Thr Val Ala Val Asp Gly Thr 420 425 430 Leu Thr Leu Ser Cys Val Ala Thr Gly Ser Pro Val Pro Thr Ile Leu 435 440 445 Trp Arg Lys Asp Gly Val Leu Val Ser Thr Gln Asp Ser Arg Ile Lys 450 455 460 Gln Leu Glu Ser Gly Val Leu Gln Ile Arg Tyr Ala Lys Leu Gly Asp 465 470 475 480 Thr Gly Arg Tyr Thr Cys Thr Ala Ser Thr Pro Ser Gly Glu Ala Thr 485 490 495 Trp Ser Ala Tyr Ile Glu Val Gln Glu Phe 500 505 <210> 27 <211> 225 <212> PRT <213> Brown rat (Rattus norvegicus) <400> 27 Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu 1 5 10 15 Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val 20 25 30 Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu 35 40 45 Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys 50 55 60 Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg 65 70 75 80 Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly 85 90 95 Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Ile Cys Val Ala Arg Asn 100 105 110 Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile 115 120 125 Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val 130 135 140 Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu 145 150 155 160 Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp 165 170 175 Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg 180 185 190 Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly 195 200 205 Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser 210 215 220 Phe 225 <210> 28 <211> 200 <212> PRT <213> Rattus norvegicus <400> 28 Phe Thr Ser Leu His Phe Val Ser Glu Pro Ser Asp Ala Val Thr Met 1 5 10 15 Arg Gly Gly Asn Val Leu Leu Asn Cys Ser Ala Glu Ser Asp Arg Gly 20 25 30 Val Pro Val Ile Lys Trp Lys Lys Asp Gly Leu Ile Leu Ala Leu Gly 35 40 45 Met Asp Asp Arg Lys Gln Gln Leu Pro Asn Gly Ser Leu Leu Ile Gln 50 55 60 Asn Ile Leu His Ser Arg His His Lys Pro Asp Glu Gly Leu Tyr Gln 65 70 75 80 Cys Glu Ala Ser Leu Gly Asp Ser Gly Ser Ile Ile Ser Arg Thr Ala 85 90 95 Lys Val Met Val Ala Gly Pro Leu Arg Phe Leu Ser Gln Thr Glu Ser 100 105 110 Ile Thr Ala Phe Met Gly Asp Thr Val Leu Leu Lys Cys Glu Val Ile 115 120 125 Gly Asp Pro Met Pro Thr Ile His Trp Gln Lys Asn Gln Gln Asp Leu 130 135 140 Asn Pro Ile Pro Gly Asp Ser Arg Val Val Val Leu Pro Ser Gly Ala 145 150 155 160 Leu Gln Ile Ser Arg Leu Gln Pro Gly Asp Ser Gly Val Tyr Arg Cys 165 170 175 Ser Ala Arg Asn Pro Ala Ser Thr Arg Thr Gly Asn Glu Ala Glu Val 180 185 190 Arg Ile Leu Ser Asp Pro Gly Leu 195 200 <210> 29 <211> 380 <212> PRT <213> Brown rat (Rattus norvegicus) <400> 29 Phe Thr Ser Leu His Phe Val Ser Glu Pro Ser Asp Ala Val Thr Met 1 5 10 15 Arg Gly Gly Asn Val Leu Leu Asn Cys Ser Ala Glu Ser Asp Arg Gly 20 25 30 Val Pro Val Ile Lys Trp Lys Lys Asp Gly Leu Ile Leu Ala Leu Gly 35 40 45 Met Asp Asp Arg Lys Gln Gln Leu Pro Asn Gly Ser Leu Leu Ile Gln 50 55 60 Asn Ile Leu His Ser Arg His His Lys Pro Asp Glu Gly Leu Tyr Gln 65 70 75 80 Cys Glu Ala Ser Leu Gly Asp Ser Gly Ser Ile Ile Ser Arg Thr Ala 85 90 95 Lys Val Met Val Ala Gly Pro Leu Arg Phe Leu Ser Gln Thr Glu Ser 100 105 110 Ile Thr Ala Phe Met Gly Asp Thr Val Leu Leu Lys Cys Glu Val Ile 115 120 125 Gly Asp Pro Met Pro Thr Ile His Trp Gln Lys Asn Gln Gln Asp Leu 130 135 140 Asn Pro Ile Pro Gly Asp Ser Arg Val Val Val Leu Pro Ser Gly Ala 145 150 155 160 Leu Gln Ile Ser Arg Leu Gln Pro Gly Asp Ser Gly Val Tyr Arg Cys 165 170 175 Ser Ala Arg Asn Pro Ala Ser Thr Arg Thr Gly Asn Glu Ala Glu Val 180 185 190 Arg Ile Leu Ser Asp Pro Gly Leu His Arg Gln Leu Tyr Phe Leu Gln 195 200 205 Arg Pro Ser Asn Val Ile Ala Ile Glu Gly Lys Asp Ala Val Leu Glu 210 215 220 Cys Cys Val Ser Gly Tyr Pro Pro Pro Ser Phe Thr Trp Leu Arg Gly 225 230 235 240 Glu Glu Val Ile Gln Leu Arg Ser Lys Lys Tyr Ser Leu Leu Gly Gly 245 250 255 Ser Asn Leu Leu Ile Ser Asn Val Thr Asp Asp Asp Ser Gly Thr Tyr 260 265 270 Thr Cys Val Val Thr Tyr Lys Asn Glu Asn Ile Ser Ala Ser Ala Glu 275 280 285 Leu Thr Val Leu Val Pro Pro Trp Phe Leu Asn His Pro Ser Asn Leu 290 295 300 Tyr Ala Tyr Glu Ser Met Asp Ile Glu Phe Glu Cys Ala Val Ser Gly 305 310 315 320 Lys Pro Val Pro Thr Val Asn Trp Met Lys Asn Gly Asp Val Val Ile 325 330 335 Pro Ser Asp Tyr Phe Gln Ile Val Gly Gly Ser Asn Leu Arg Ile Leu 340 345 350 Gly Val Val Lys Ser Asp Glu Gly Phe Tyr Gln Cys Val Ala Glu Asn 355 360 365 Glu Ala Gly Asn Ala Gln Ser Ser Ala Gln Leu Ile 370 375 380 <210> 30 <211> 71 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 30 cgcgtccccg ccgccacatt tcgtggtaaa ttcaagagat ttacaacgaa atgtggcggc 60 tttttggaaa t 71 <210> 31 <211> 69 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 31 cgatttccaa aaagccgcca catttcgttg taaatctctt gaatttacaa cgaaatgtgg 60 cggcgggga 69 <210> 32 <211> 71 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 32 cgcgtccccc tgcgataatg cgaggaagat ttcaagagaa tcttcctctc attatcgcag 60 tttttggaaa t 71 <210> 33 <211> 69 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 33 cgatttccaa aaactgcgat aatgcgagga agattctctt gaaatcttcc tcgcattatc 60 gcaggggga 69 <210> 34 <211> 86 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 34 cgcgtccccc cattcgctct gtagtaatag gtggctgttc aagagacaag ccacctatta 60 ctacagagcg aatggttttt ggaaat 86 <210> 35 <211> 85 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 35 cgatttccaa aaaccattcg ctctgtagta ataggtggct tgtctcttga acaagccacc 60 tattactaca gagccaatgg gggga 85 <210> 36 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 36 gtgggtccta acgcagtgtc 20 <210> 37 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 37 acaaaggcgc aatccaatat g 21 <210> 38 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 38 tgagctgaga aggctggtac 20 <210> 39 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 39 accccaaact ccgatagtcc 20 <210> 40 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 40 ccgcatcaac agtagccttt 20 <210> 41 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 41 tgcaagacct cagctttctc 20 <210> 42 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 42 tctgccaaac caacgaggag tg 22 <210> 43 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 43 agaagccagc tttcggaaca cc 22 <210> 44 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 44 gagtcggaga acatctgtgg ca 22 <210> 45 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 45 cttctcagag cacatgggct tg 22 <210> 46 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 46 gctcttcgtg agcaccagaa c 21 <210> 47 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 47 ccacccattc ttttcactcg gac 23 <210> 48 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 48 agacaacgac gacttcgagg ag 22 <210> 49 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 49 gtaccatcca gaggaggtgc aac 23 <210> 50 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 50 ttatggtgat gtggacctta gta 23 <210> 51 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 51 ggttgtatgg gatggttgga g 21 <210> 52 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 52 accctgcctt tgagcatcag ac 22 <210> 53 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 53 gcttgtactg gtcgcagcag aa 22 <210> 54 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 54 catggccttc cgtgttccta 20 <210> 55 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 55 cctgcttcac caccttcttg at 22

Claims

1. A non-therapeutic method for promoting milk production in mammals, the method comprising: The administration of the soluble extracellular domain of mammalian ROBO1 to the mammal promoted milk production.

2. The method according to claim 1, wherein the soluble mammalian ROBO1 extracellular domain is a soluble rodent, bovine, sheep, goat, camel, or human ROBO1 extracellular domain.

3. The method according to claim 1, wherein the mammal is a cow and the soluble mammalian ROBO1 extracellular domain is a soluble bovine ROBO1 extracellular domain.

4. The method according to any one of claims 1-3, wherein the soluble mammalian ROBO1 extracellular domain is fused with a heterologous polypeptide, and wherein the heterologous polypeptide is a His tag, a hemagglutinin tag, a human or murine immunoglobulin (Ig) Fc region, or a Myc tag.

5. The method according to claim 4, wherein the heterologous polypeptide is the Fc region of human or mouse immunoglobulin (Ig).

6. Use of a soluble mammalian ROBO1 extracellular domain fused with a heterologous peptide to prepare a drug for promoting milk production in mammals, wherein the heterologous peptide is a His tag, a hemagglutinin tag, a human or mouse Fc region, or a Myc tag.

7. The use according to claim 6, wherein the soluble mammalian ROBO1 extracellular domain is a soluble rodent, bovine, sheep, goat, camel, or human ROBO1 extracellular domain.

8. The use according to claim 6 or 7, wherein the mammal is a bovine and the soluble mammalian ROBO1 extracellular domain is a soluble bovine ROBO1 extracellular domain.

9. The use according to claim 6, wherein the heterologous polypeptide is a human or mouse Fc region.

10. A transgenic mammalian mammary gland tissue cell containing a gene modification that results in the expression of the extracellular domain of soluble mammalian ROBO1.

11. The transgenic mammalian mammary gland tissue cells according to claim 10, wherein the transgenic mammalian mammary gland tissue cells are transgenic bovine, sheep, goat, or camel mammary gland tissue cells.

12. The transgenic mammalian mammary gland tissue cell according to claim 10 or 11, wherein the transgenic mammalian mammary gland tissue cell is a transgenic bovine mammary gland tissue cell and the soluble mammalian ROBO1 extracellular domain is a soluble bovine ROBO1 extracellular domain.

13. A method for generating a transgenic mammal comprising a gene modification that causes expression of a soluble mammalian ROBO1 extracellular domain in mammary tissue, the method comprising: 1) Gene constructs containing nucleic acids encoding the extracellular domain of soluble mammalian ROBO1 under the control of a mammary gland-specific promoter; 2) Transfect the gene construct into cells from mammals and select transgenic cells that have incorporated the gene construct; 3) Fuse the transgenic cell with an enucleated oocyte from the same species as the transgenic cell and allow the oocyte to develop into an embryo; 4) Transfer the embryo into a recipient mammal of the same species as the embryo; 5) Confirm that the embryo develops into a transgenic mammal.

14. The method of claim 13, wherein the transgenic mammal is a cow, sheep, goat, or camel.

15. The method according to claim 13 or 14, wherein the transgenic mammal is a bovine and the soluble mammalian ROBO1 extracellular domain is a soluble bovine ROBO1 extracellular domain.

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