Genetically modified mice for selective depletion of antibody-secreting cells

Abstract

The disclosure relates to genetically modified non-human animals, including mice, in which a diphtheria toxin receptor (DTR) gene is inserted into the endogenous Jchain locus. Also provided are methods of making the animals, and uses of the animals for selective deletion of antibody-secreting cells (ASCs).

Description

GENETICALLY MODIFIED MICE FOR SELECTIVE DEPLETION OF ANTIBODY-SECRETING CELLSCross-Reference to Related Application

[0001] This application claims the benefit of priority to United States Provisional Application No. 63 / 568,498 filed March 22, 2024, the contents of which are incorporated herein by reference in their entirety.Field

[0002] The present disclosure relates to genetically modified non-human animals and cells in which exogenous diphtheria toxin receptor (DTR) is inserted into the endogenous Jchain locus, methods of making the animals and cells, and uses thereof.Background

[0003] Antibody-secreting cells (ASCs) are generated following B cell activation and constitutively secrete antibodies. ASCs are key mediators of humoral immunity whether it be in the context of pathogen exposure, vaccination or even homeostatic clearance of cellular debris. As such, the ability to selectively deplete ASCs in a wide variety of experimental settings is valuable in understanding their biological roles.

[0004] Genetic models have been developed to fluorescently timestamp ASCs which have led to new insights regarding ASC longevity as well as the developmental relationship between short-lived plasmablasts (PBs) and post-mitotic plasma cells (PCs) [7,9-11], Many of these experiments have been performed in the context of a fully replete ASC compartment constraining the ability to assess ASC longevity in competitive versus non-competitive scenarios. While efforts have been made to subvert these issues, these experiments largely focused on the maintenance of pre-existing ASCs. While pre-existing ASCs are important as they can represent a decades long record of immunization and humoral protection, understanding how newly formed ASCs integrate into a long-lived protective reservoir is essential especially considering the recent COVID-19 pandemic and continual threat of newly emerging viruses. Along these lines, being able to assess ASC generation in the presence or absence of pre-existing ASCs is very informative. Towards this goal, CD138 Abs have been shown to be useful in depleting ASCs in bone marrow (BM) of young and old mice

[0012] , However, the CD138 Abs utilized in those studies were a custom mouse anti-mouse versionof the commercial clone 281-2 and would require in house production

[0012] , The commercially available version of clone 281-2 was shown to promote mobilization of myeloma cells (i.e. , plasma cell cancer) but was unable to induce cellular depletion unless paired with bortezomib, an NF-KB inhibitor

[0013] , CD138-diphtheria toxin receptor (DTR) mice have also been generated and utilized in the context of Plasmodium infection

[0014] , Taken together, the CD138-driven models have inherent limitations. For example, the differential level of Cd138 expression compared to other selected immune cell types is not particularly high

[0011] suggesting a narrow window allowing for ASC depletion with limited off target effects. Recently, the BICREAD mouse strain was developed which incorporates both a Tamoxifen- inducible Cre recombinase (CreERT2) and DTR within the Prdml locus [7], While effective regarding ASC depletion, the use of Prdml to drive DTR expression presents complications given its role in regulating both CD4 and CD8 memory T cell formation [15-17],

[0005] Accordingly, new model systems and research tools for depleting ASCs are desired, in particular tools which allow users to modulate the presence of pre-existing ASCs.Summary

[0006] Genetically modified mice expressing the diphtheria toxin receptor (DTR) in the Jchain gene locus, thereby driving DTR expression in antibody-secreting cells (ASCs), were prepared.

[0007] Accordingly, the present disclosure provides a cell of a genetically modified non-human animal, wherein the genome of the genetically modified non-human animal comprises an exogenous diphtheria toxin receptor (DTR) gene inserted in the endogenous Jchain gene of the non-human animal.

[0008] In one embodiment, the wild-type animal is insensitive to diphtheria toxin.

[0009] In another embodiment the animal is a mouse or a rat. In a further embodiment, the animal is a mouse.

[0010] In one embodiment, the DTR gene is a human or simian DTR gene.

[0011] In one embodiment, the DTR gene is inserted into an exon of the Jchain gene.Optionally, the exon is exon 4. Optionally, the DTR gene is inserted into the 3’IITR of exon 4 of the Jchain gene.

[0012] In one embodiment, the DTR gene comprises SEQ ID NO: 1 or a functional fragment thereof, or a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to SEQ ID NO: 1 or a functional fragment thereof.

[0013] In one embodiment, the cell is an embryonic stem cell.

[0014] The disclosure further provides a genetically modified mouse or rat embryonic stem (ES) cell, wherein the genome of the ES cell comprises an exogenous diphtheria toxin receptor (DTR) gene inserted in the endogenous Jchain gene.

[0015] In one embodiment, the DTR gene is a human or simian DTR gene.

[0016] In another embodiment, the DTR gene is inserted into an exon of the Jchain gene. Optionally, the exon is exon 4. Optionally, the DTR gene is inserted into the 3’IITR of exon 4 of the Jchain gene.

[0017] In another embodiment, the DTR gene comprises SEQ ID NO: 1 ora functional fragment thereof, or a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to SEQ ID NO: 1 or a functional fragment thereof.

[0018] The disclosure further provides a genetically modified non-human animal whose genome comprises a diphtheria toxin receptor (DTR) gene inserted in the Jchain gene of the non-human animal.

[0019] In one embodiment, the wild-type animal is insensitive to diphtheria toxin.

[0020] In another embodiment, the animal is a mouse or a rat.

[0021] In another embodiment, the animal is a mouse.

[0022] In another embodiment, the DTR gene is a human or simian DTR gene.

[0023] In another embodiment, DTR gene is inserted into an exon of the Jchain gene.Optionally, the exon is exon 4. Optionally, the DTR gene is inserted into the 3’IITR of exon 4 of the Jchain gene.

[0024] In another embodiment, the DTR gene comprises SEQ ID NO: 1 ora functional fragment thereof, or a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to SEQ ID NO: 1 or a functional fragment thereof.

[0025] In another embodiment, the animal expresses Jchain protein at a level of at least 50%, 60%, 70%, 80%, 90% or 95% of the level that a wild type animal expresses Jchain protein.

[0026] In another embodiment, the animal expresses DTR in antibody-secreting cells (ASCs).

[0027] The disclosure also provides method of making a genetically modified nonhuman animal or animal cell comprising modifying a genome of a non-human animal or animal cell to comprise a DTR gene, wherein the DTR gene is inserted in the Jchain gene of the non-human animal, and generating a non-human animal or animal cell comprising the modified genome.

[0028] In one embodiment, the wild-type animal is insensitive to diphtheria toxin.

[0029] In another embodiment, said modifying comprises introducing a nucleic acid molecule comprising a DTR nucleic acid sequence into the genome of a non-human animal embryonic stem (ES) cell, obtaining a non-human animal ES cell in which the DTR nucleic acid sequence has been inserted into an exon of the endogenous Jchain gene, and generating an animal from the obtained non-human animal ES cell.

[0030] In another embodiment, the animal is a mouse or rat.

[0031] In another embodiment, the animal is a mouse.

[0032] In another embodiment, the DTR gene is inserted into an exon of the Jchain gene. Optionally the exon is exon 4. Optionally, the DTR gene is inserted into the 3’IITR of exon 4 of the Jchain gene.

[0033] In another embodiment, the DTR gene comprises SEQ ID NO: 1 or a functional fragment thereof, or a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to SEQ ID NO: 1 or a functional fragment thereof.

[0034] In another embodiment, the animal expresses DTR in antibody-secreting cells (ASCs).

[0035] The disclosure further provides a method of depleting ASCs in a non-human animal comprising administering diphtheria toxin to a genetically modified animal as described herein.

[0036] In one embodiment, the method comprises a single administration of diphtheria toxin.

[0037] The disclosure further provides use of a genetically modified animal as described herein for assessing differentiation kinetics of antibody secreting cells (ASCs).

[0038] The disclosure further provides use of a genetically modified animal as described herein for assessing the level of antibody secreting cells (ASCs) in a tissue or organ of interest.

[0039] The disclosure further relates to the use of the genetically modified animals of the disclosure to study and design therapies for diseases involving expression of antibodysecreting cells.

[0040] These and other features and advantages of the present disclosure will become apparent from the following detailed description taken together with the accompanying drawings. It should be understood, however, that the detailed description and specific examples, while indicating preferred implementations of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those of skill in the art from this detailed description.Brief Description of the Drawings

[0041] For a better understanding of the various embodiments described herein, and to show more clearly how these various embodiments may be carried into effect, reference will be made, by way of example, to the accompanying drawings which show at least one example embodiment, and which are now described. The drawings are not intended to limit the scope of the teachings described herein.

[0042] FIG. 1 shows construction and genotyping of Jc / ia / n-Diphtheria Toxin Receptor (DTR) mice (J-DTR). (A) Schematic showing wildtype (WT) Jchain locus, the Targeting Construct and the final Jchain-DTR targeted insertion following Neomycin resistance (NeoR) cassette deletion by Flippase (FLP). Schematic is drawn to approximate scale. F = FRT sites used for FLP-mediated recombination, IRES = internal ribosomal entry site. 5’ and 3’ homology arms used to direct integration are shown. Large bent arrows indicate direction of transcription for DTR and NeoR. Small arrow heads indicate approximate placement for genotyping primers. Black arrow heads bind DNA regions present in the endogenous Jchaingene. Blue arrowhead binds DNA sequence within the DTR coding region. Note that following NeoRdeletion, a single FRT site is reconstituted. This site is omitted for clarity. (B) Representative Jchain-DTR genotyping. Note that the 434 bp WT product is only observed in animals lacking the IRES-DTR insertion. Animals containing this insertion would generate a product approximating 1664 bp which is not amplified using the current PCR conditions. (C) Representative FLP genotyping. (B-C) All PCR products were amplified from genomic DNA and electrophoresed in a 2% agarose gel containing ethidium bromide. The 1 kb+ DNA ladder was utilized as a size standard.

[0043] FIG. 2 shows the validation of DTR gene expression by antibody-secreting cells (ASCs) from Jchain-DTR mice. (A) Representative flow cytometry pseudocolor plots showing gating of CD19+CD138- / LoB cells and CD138HiCD267(TACI)+ASCs in total spleen (SPL) or cells purified using STEMCELL Technologies Pan-B and ASC isolation kits. Numbers in plots indicate percentages of gated populations within total live singlets. (B) Quantification of ASC percentages in total SPL or cells purified using Pan-B and ASC isolation kits. (C-E) Relative gene expression of (C) Prdml, (D) Jchain and (E) DTR (HBEGF) in cells purified using Pan-B and ASC isolation kits. All values are relative to the expression of Actb. (F) DTR expression normalized to Prdml expression in WT and DTR ASCs. (B-F) Symbols represent individual 2- 4 months old female (orange) and male (blue) mice. Horizontal lines represent mean ± standard error of the mean (SEM). Statistics: (B) One-way ANOVA with Dunnett’s multiple comparisons test with ASCs set as the control column. (C-F) Unpaired Student’s t-test.

[0044] FIG. 3 shows the validation of DTR surface protein expression by ASCs from Jchain-DTR mice. (A) Representative flow cytometry pseudocolor plots showing gating of SPL ASCs as CD138HilgD / LoCD90.2- / LoCD267(TACI)+CD44+. ASC subset gating is shown for P1 (CD45R(B220)+CD19+), P2 (CD45R(B220)- CD19+) and P3 (CD45R(B220)- CD19’). Numbers in plots indicate percentages of gated populations within the immediate parent population. (B) Representative flow cytometry histogram overlays showing surface expression of DTR by SPL ASCs and B cells from both WT and Jchain-DTR mice. SPL B cells were gated as CD19+CD138' / Lo. Numbers in plots indicate DTR geometric mean fluorescence intensities (gMFI). (C- E) gMFI for WT and Jchain-DTR ASCs from (C) SPL, (D) bone marrow (BM) and (E) thymus (THY). (F-H) DTR gMFI for WT and Jchain-DTR ASC P1-P3 subsets from (F) SPL, (G) BM and (H) THY. (C-H) Symbols represent individual 2-4 months old female (orange) and male (blue)mice. Horizontal lines represent mean ± standard error of the mean (SEM). Statistics: (C-E) Unpaired Student’s t-test. (F-H) One-way ANOVA with Tukey’s multiple comparisons test.

[0045] FIG. 4 shows that Jchain-DTR mice demonstrate acute ASC depletion following a single dose of diphtheria toxin (DT). (A) Schematic showing DT treatment of WT and Jchain- DTR mice. Animals were given a single intraperitoneal dose of 200 ng DT in 100 pL 1x phosphate buffered saline (PBS). Control mice received 100 pL of 1x PBS. Mice were euthanized following 1 day and bone marrow (BM), spleen (SPL) and thymus (THY) were assessed for ASCs via flow cytometry. (B) Representative flow cytometry pseudocolor plots showing gating of SPL ASCs from DTR treated with 1x PBS or DT. Numbers in plots indicate percentages of gated populations within the immediate parent population. Cells were initially gated on live singlets. (C-E) Total cell numbers for (C) BM, (D) SPL, and (E) THY of WT and DTR mice treated with PBS or DT. (F-H) Total ASC numbers for (F) BM, (G) SPL, and (H) THY of WT and DTR mice treated with PBS or DT. (I-K) Percentages of P1-P3with ASC populations from (I) BM, (J) SPL, and (K) THY of DTR mice treated with PBS or DT. (C-K) Symbols represent individual 2-4 months old female (orange) and male (blue) mice. Horizontal lines represent mean ± standard error of the mean (SEM). Statistics: Unpaired Student’s t-test with comparisons made between (C-H) PBS and DT treatments for each genotype or (l-K) PBS and DT treatments for each ASC subpopulation.

[0046] FIG. 5 shows a PCR amplification performed to detect presence of the knockin IRES cassette.

[0047] FIG. 6 shows a PCR amplification using primer set newFLPI and newFLP2 was used to screen mice for the FLP transgene.

[0048] FIG. 7 shows a PCR amplification using primer set PNDEL1 and PNDEL2 was used to screen mice for the deletion of the Neo cassette.

[0049] FIG. 8 shows a PCR amplification of tail DNA samples from positive mice with primers NEOGT and A2.

[0050] FIG. 9 shows genotyping of J-DTR mice. Representative Jchain-DTR genotyping results generated from PCR amplification of genomic DNA. Note that the 434 bp WT product is only observed in animals lacking the IRES-DTR insertion. Animals containing this insertion would generate a product approximating 1664 bp which is not amplified usingthe current PCR conditions. PCR products were electrophoresed in a 2% agarose gel containing ethidium bromide. A 1 kb+ DNA ladder (L) was utilized as a size standard.

[0051] FIG. 10 shows validation of DTR gene expression by ASCs from J-DTR mice.(A) Quantification of ASC percentages in total SPL or purified cells using Pan-B and ASC isolation kits. (B-D) Relative gene expression of (B) Prdml , (C) Jchain and (D) DTR (HBEGF) in cells purified using Pan-B and ASC isolation kits. All values are relative to the expression of Actb. (E) DTR expression normalized to Prdml expression in WT and J-DTR ASCs. (F) Jchain expression normalized to Prdml expression in WT and J-DTR ASCs. (A-F) Symbols represent individual 3-7 months old female (orange) and male (blue) mice. Horizontal lines represent mean ± standard error of t he mean (SEM). WT SPL, Pan-B and ASC: female n = 2, male n = 3; J-DTR SPL, Pan-B and ASC: female n = 3, male n = 3. (A) Statistics: One-way ANOVA with Dunnett’s multiple comparisons test with ASCs for each genotype set as the control column. (B-E) Statistics: U npaired Student’s t-test.

[0052] FIG. 11 shows validation of DTR surface protein expression by ASCs from J- DTR mice. (A) Representative flow cytometry pseudocolor plots showing gating of SPL ASCs as CD138HIlgD / LOCD90.2 / LOCD267(TACI)+CD44+. ASC subset gating is shown for PBs (CD45R(B220)+) and PCs (CD45R(B220) ). Numbers in plots indicate percentages of gated populations within the immediate parent population. (B) Representative flow cytometry histogram overlays showing surface expression of DTR by SPL ASCs and B cells from both WT and J-DTR mice. SPL B cells were gated as CD19+CD138 / LO. Numbers in plots indicate DTR gMFIs. (C-E) gMFIs for WT and J-DTR ASCs from (C) SPL, (D) BM and (E) THY. (F- H) DTR gMFIs for WT and J-DTR PBs and PCs from (F) SPL, (G) BM and (H) THY. (C-H) Symbols represent individual 3-7 months old female (orange) and male (blue) mice. Horizontal lines represent mean ± SEM. WT: female n = 8, male n = 9; J-DTR: female n = 10, male n = 10. (C-E) Statistics: Unpaired Student’s t-test. (F-H) Statistics: Unpaired Student’s t-test comparing WT and J-DTR samples. Paired Student’s t-test comparing PBs and PCs within a genotype.

[0053] FIG. 12 shows ASCs develop normally in the SPL, BM and THY of J-DTR mice. (A-C) Total ASC numbers for (A) SPL, (B) BM and (C) THY of WT and J-DTR mice. (D) Representative ELISpot images showing IgM, IgG and IgA spot formation from SPL, BM and THY of WT and J-DTR mice. One hundred thousand (105) cells per well were plated in triplicate. Numbers indicate spots counted per well following background subtraction of identical wells not coated with capture Ab. (E-G) Numbers of (E) IgM, (F) IgG and (G) IgA spots per 105cells from SPL, BM and THY of WT and J-DTR mice. (H-J) Plasma concentration of (H) IgM, (I) IgG and (J) IgA from WT and J-DTR mice. (K) Fecal IgA concentration from WT and J-DTR mice. (A-C) Symbols represent individual 3-7 months old female (orange) and male (blue) mice. Horizontal lines represent mean ± SEM. WT: female n = 8, male n = 10; J-DTR: female n = 10, male n = 11. (E-G) Symbols represent individual wells plated in triplicate from 3-5 months old female (orange) and male (blue) WT and J-DTR mice. Values shown represent data following subtraction of spots detected in wells that were not coated with capture Ab (background). Horizontal lines represent mean ± SEM. WT: female n = 4, male n = 5. J-DTR: female n = 5, male n = 6. (H-K) Symbols represent individual 3-5 months old female (orange) and male (blue) WT and J-DTR mice. Horizontal lines represent mean ± SEM. WT: female n = 4, male n = 5. J-DTR: female n = 5, male n = 6. Statistics: Unpaired Student’s t-test.

[0054] FIG. 13 shows single dose administration of DT leads to the acute depletion of ASCs in J-DTR mice. (B) Representative flow cytometry pseudocolor plots showing gating of SPL ASCs from J-DTR mice treated with PBS or DT. Cells were initially gated on live singlets and numbers in plots indicate percentages of ASCs within total live singlets. (C-E) Total cell numbers for (C) SPL, (D) BM and (E) THY of WT and J-DTR mice treated with PBS or DT. Data presented on Log 10 scale to show full range. (F-H) Total ASC numbers for (F) SPL, (G) BM and (H) THY of WT and J-DTR mice treated with PBS or DT. Data presented on Log10 scale to show full range. (I-K) PB and PC numbers for (I) SPL, (J) BM and (K) THY of J-DTR mice treated with PBS or DT. Data presented on Logw scale to show full range. Values of zero are not visible using a log scale. (C-K) Symbols represent individual female (orange) and male (blue) mice. Horizontal lines represent mean ± SEM. (C-H) WT PBS: female n = 3, male n = 5; WT DT: female n = 4, male n = 5; J-DTR PBS: female n = 8, male n = 8; J-DTR DT: female n = 8, male n = 9. Statistics: Unpaired Student’s t-test with comparisons made between PBS and DT treatments within a genotype. (I-K) J-DTR PBS:female n = 8, male n = 8; J-DTR DT: female n = 8, male n = 9. Statistics: Unpaired Student’s t-test with comparisons made between PBS and DT treatments within an ASC subset.

[0055] FIG. 14 shows single dose administration of DT leads to the loss of Ab spot formation and fecal IgA in J-DTR mice. (A) Representative ELISpot images showing IgM, IgG and IgA spot formation from SPL of WT and J-DTR mice treated with PBS or DT. One hundred thousand (105) cells per well were plated in triplicate. Numbers indicate spots counted per well following background subtraction of identical wells not coated with capture Ab. (B-D) Numbers of IgM spots per 105cells from (B) SPL, (C) BM and (D) THY of WT and J-DTR mice treated with PBS or DT. (E-G) Numbers of IgG spots per 105cells from (E) SPL, (F) BM and (G) THY of WT and J-DTR mice treated with PBS or DT. (H-J) Numbers of IgA spots per 105cells from (H) SPL, (I) BM and (J) THY of WT and J-DTR mice treated with PBS or DT. (K) Fecal IgA concentration from WT and J-DTR mice treated with PBS or DT. (B-J) Symbols represent individual wells plated in triplicate from 3-5 months old female (orange) and male (blue) WT and J-DTR mice. Values shown represent data following subtraction of spots detected in wells that were not coated with capture Ab (background). Some symbols overlap due to having the same value (e.g., 0). Horizontal lines represent mean ± SEM. WT PBS: female n = 1 , male n = 2; WT DT: female n = 2, male n = 1 ; J-DTR PBS: female n = 2, male n = 2; J-DTR DT: female n = 1 , male n = 3. Statistics: Unpaired Student’s t-test with comparisons made between PBS and DT treatments within a genotype. (K) Symbols represent individual 3-5 months old female (orange) and male (blue) WT and J- DTR mice. Horizontal lines represent mean ± SEM. WT PBS: female n = 1 , male n = 2; WT DT: female n = 2, male n = 2; J-DTR PBS: female n = 3, male n = 2; J-DTR DT: female n = 3, male n = 3. Statistics: Unpaired Student’s t-test with comparisons made between PBS and DT treatments within a genotype.

[0056] FIG. 15 shows single dose DT administration allows for the assessment of ASC reconstitution kinetics in J-DTR mice. (A) Schematic showing DT treatment of J-DTR mice. 3-4 months old animals were given a single i.p. dose of 200 ng DT in 100 mL 1x PBS. Control mice received 100 mL of 1x PBS. Mice were euthanized at days 1 , 3 and 7 post-injection. SPL, BM and THY were assessed for ASCs via flow cytometry. Schematic made with BioRender. (B-D) Total cell numbers for (B) SPL, (C) BM and (D) THY of J-DTR mice treated with PBS or DT. Data presented on Logw scale to show full range. (E-G) Total ASC numbers for (E) SPL, (F) BM and (G) THY of J-DTR mice treated with PBS or DT. Data presented onLog10 scale to show full range. (H-J) DTR gMFIs for ASCs from (H) SPL, (I) BM and (J) THY of J-DTR mice treated with PBS or DT. (B-J) Symbols represent individual female (orange) and male (blue) mice. Horizontal lines represent mean ± SEM. J-DTR day 1 PBS: female n = 2, male n = 2; J-DTR day 1 DT: female n = 2, male n = 1 ; J-DTR day 3 PBS: female n = 2, male n = 2; J-DTR day 3 DT: female n = 1 , male n = 2; J-DTR day 7 PBS: female n = 2, male n = 2; J-DTR day 7 DT: female n = 2, male n = 2. (B-G) Statistics: Kruskal-Wallis test (nonparametric) with Dunn’s multiple comparisons test. Comparisons made between PBS and DT treatments for a given day. (H-J) Statistics: One-way ANOVA with Tukey’s multiple comparisons test. Comparisons made between D1 , D3 and D7 for a given treatment.

[0057] FIG. 16 shows Jchain is highly expressed in ASCs. (A) ImmGen data showing Prdml and Sdc1 gene expression for all cell types. (B) ImmGen data showing Prdml, Sdc1 and Jchain gene expression for all cell types. (C) ImmGen data showing Prdml, Sdc1 and Jchain gene expression for all B cell subsets. (A-C) Data are derived from the ImmGen RNA- sequencing database and shown as DESeq2 processed expression levels.

[0058] FIG. 17 shows Jchain protein is expressed in J-DTR SPL ASC-enriched samples. (A) Ponceau stained membrane showing protein loading for SPL ASC-enriched samples from female (F) and (M) WT (+ / +) and J-DTR (+ / D) mice. Molecular weights for the PageRuler Plus Prestained Protein Ladder (L) are provided for reference. Auto Tone in Photoshop was applied to the Ponceau stained membrane for visualization. (B) Western blot detection of Jchain protein for SPL ASC-enriched samples. The bottom third of the membrane from (A) was cut away and separately probed with an anti-Jchain primary antibody diluted 1 :250. Subsequently, the membrane was probed with a goat anti-rabbit IgG- HRP secondary antibody diluted at 1 :10,000. The membrane was imaged on a Bio-Rad ChemiDoc using chemiluminescence detection and is presented as is.

[0059] FIG. 18 shows validation of DTR surface protein expression by mlgM+, mlgA+and DN ASCs from J-DTR mice. (A) Representative flow cytometry zebra plots showing gating of mlgM+, mlgA+and DN ASCs from SPL, BM and THY. (B) Representative flow cytometry histogram overlays showing surface expression of DTR by mlgM+, mlgA+and DN ASCs from both WT and J-DTR mice. Data from SPL, BM and THY are shown. Numbers in plots indicate DTR gMFIs. (C-E) DTR gMFIs for WT and J-DTR mlgM+, mlgA+and DN ASCs from (C) SPL, (D) BM and (E) THY. Symbols represent individual 3-5 months old female(orange) and male (blue) J-DTR mice. Horizontal lines represent mean ± SEM. J-DTR: female n = 5, male n = 6. Statistics: One-way ANOVA with Tukey’s multiple comparisons test.

[0060] FIG. 19 shows DTR surface protein expression by B cells from J-DTR mice. (A) Representative flow cytometry pseudocolor plots showing gating of SPL B cells and GCBs. (B) Representative flow cytometry histogram overlays showing surface expression of DTR by SPL CD138HICD90.2' cells and GCBs from both WT and J-DTR mice. Numbers in plots indicate DTR gMFIs. (C) Representative flow cytometry pseudocolor plots showing gating of THY B cells and GCB-like cells. (D) Representative flow cytometry histogram overlays showing surface expression of DTR by THY CD138HICD90.2' cells and GCB-like cells from both WT and J-DTR mice. Numbers in plots indicate DTR gMFIs. (E-F) DTR gMFIs for WT and J-DTR B cells, GCB (or GCB-like) and CD138HICD90.2- cells from (E) SPL and (F) THY. Symbols represent individual 3-7 months old female (orange) and male (blue) mice. Horizontal lines represent mean ± SEM. WT: female n = 4, male n = 4; J-DTR: female n = 5, male n = 4. Statistics: Unpaired Student’s t-test comparing WT and J-DTR samples.

[0061] FIG. 20 shows single dose administration of DT leads to the acute depletion of mlgM+, mlgA+and DN ASCs in J-DTR mice. (A) Schematic showing DT treatment of WT and J-DTR mice. 3-4 months old animals were given a single i.p. dose of 200 ng DT in 100 pL 1x PBS. Control mice received 100 pL of 1x PBS. Mice were euthanized the next day and SPL, BM and THY were assessed for ASC populations via flow cytometry. Schematic made with BioRender. (B-D) PB:PC ratios from (B) SPL, (C) BM and (D) THY of J-DTR mice treated with PBS or DT. Ratios calculated using absolute cell numbers. Only animals with non-zero populations for both PBs and PCs were assessed. (E-G) DTR gMFIs for ASCs from (E) SPL, (F) BM and (G) THY of WT and J-DTR mice treated with PBS or DT. (H-J) Numbers of mlgM+, mlgA+and DN ASCs from (H) SPL, (I) BM and (J) THY of J-DTR mice treated with PBS or DT. (B-J) Symbols represent individual female (orange) and male (blue) mice. Horizontal lines represent mean ± SEM. (B-D) J-DTR PBS: female n = 8, male n = 8; J-DTR DT: female n= 8, male n = 9. Statistics: Unpaired Student’s t-test. (E-G) WT PBS: female n = 3, male n = 8; WT DT: female n= 5, male n = 8; J-DTR PBS: female n = 10, male n = 10; J-DTR DT: female n = 10, male n = 11. Statistics: Unpaired Student’s t-test with comparisons made between PBS and DT treatments within a genotype. (H-J) J-DTR PBS: female n = 3, male n = 3; J-DTR DT: female n = 3, male n = 4. Statistics: Unpaired Student’s t-test.

[0062] FIG. 21 shows single dose DT administration leads to a modest SPL GCB reduction in J-DTR mice. (A) Schematic showing DT treatment of WT and J-DTR mice. 3-4 months old animals were given a single i.p. dose of 200 ng DT in 100 pL 1x PBS. Control mice received 100 pL of 1x PBS. Mice were euthanized the next day and SPL and THY B cell populations were assessed via flow cytometry. Schematic made with BioRender. (B-C) B cell numbers for (B) SPL and (C) THY of WT and J-DTR mice treated with PBS or DT. Data presented on Log10 scale to show full range. (D-E) GCB (or GCB-like) numbers for (D) SPL and (E) THY of WT and J-DTR mice treated with PBS or DT. Data presented on Log10 scale to show full range. (B-E) Symbols represent individual female (orange) and male (blue) mice. Horizontal lines represent mean ± SEM. WT PBS and DT: female n = 2, male n = 2; J- DTR PBS and DT: female n = 5, male n = 5. Statistics: Unpaired Student’s t-test with comparisons made between PBS and DT treatments within a genotype.

[0063] FIG. 22 shows single dose DT administration results in transiently reduced SPL GCBs in J-DTR mice. (A) Schematic showing DT treatment of J-DTR mice. 3-4 months old mice were given a single i.p. dose of 200 ng DT in 100 pL 1x PBS. Control mice received 100 pL of 1x PBS. Mice were euthanized at days 1 , 3 and 7 post-injection. SPL, BM and THY were assessed for B cell populations via flow cytometry. Schematic made with BioRender. (B-C) B cell numbers for (B) SPL and (C) THY of J-DTR mice treated with PBS or DT. Data presented on Log10 scale to show full range. (D-E) GCB (or GCB-like) numbers for (D) SPL and (E) THY of J-DTR mice treated with PBS or DT. Data presented on Log10 scale to show full range. (B-E) Symbols represent individual female (orange) and male (blue) mice. Horizontal lines represent mean ± SEM. J-DTR day 1 PBS: female n = 2, male n = 2; J-DTR day 1 DT: female n = 2, male n = 1 ; J-DTR day 3 PBS: female n = 2, male n = 2; J-DTR day 3 DT: female n = 1 , male n = 2; J-DTR day 7 PBS: female n = 2, male n = 2; J-DTR day 7 DT: female n = 2, male n = 2. Statistics: Kruskal-Wallis test (nonparametric) with Dunn’s multiple comparisons test. Comparisons made between PBS and DT treatments for a given day.

[0064] Further aspects and features of the example embodiments described herein will appear from the following description taken together with the accompanying drawings.Detailed Description of the Disclosure

[0065] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art.

[0066] All features disclosed in the specification, including the claims, abstract, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise.L Definitions

[0067] In understanding the scope of the present disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

[0068] The term “consisting” and its derivatives, as used herein, are intended to be closed ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps.

[0069] Further, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0070] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise.

[0071] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e. , elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified.

[0072] As used herein, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of' or "exactly one of" or, when used in the claims, "consisting of" will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0073] As used herein, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.

[0074] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0075] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0076] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0077] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, examples of methods and materials are now described.I. Genetically modified animals and cells

[0078] The present inventors generated a mouse model in which simian HBEGF (a.k.a. DTR) cDNA from Chlorocebus sabaeus was inserted into the endogenous Jchain locus thus targeting DTR expression to ASCs (referred to herein as J-DTR mice). It was shown that these mice are functional, and that IgM, IgG and IgA ASCs can be acutely depleted following a single dose of diphtheria toxin (DT) in organs such as the spleen (SPL), BM and thymus (THY). Furthermore, due to the short half-life of DT, the utility of this model in being able to assess ASC differentiation kinetics following depletion was demonstrated. At homeostasis, ASC populations were reconstituted to normal levels 7 days following DT injection supporting the concept that ASCs are continuously produced even in the absence of overt infection.

[0079] Accordingly, the present disclosure provides a genetically modified non-human animal or animal cell whose genome comprises an exogenous DTR gene inserted in the endogenous Jchain gene of the non-human animal.

[0080] The term “genetically modified non-human animal” includes any member of the animal kingdom, except humans, in which one or more cells contain a genetic modification introduced by way of human intervention such as by genetic modification techniques knownin the art. For example, an exogenous nucleic acid may be introduced into the cell, directly or indirectly, by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The nucleic acid may be integrated within a chromosome. Non-limiting examples of animals that may be used in the present disclosure include mice, rats, squirrels, hamsters, guinea pigs, rabbits, pigs, sheep, baboons, monkeys, chimpanzees, birds and amphibians. In one embodiment, the animal is a mammal.

[0081] In one embodiment, the animal is an animal which is insensitive to diphtheria toxin. Examples of animals which are insensitive to diphtheria toxin include mice and rats. Thus, in one embodiment, the animal is a mouse or a rat. In another embodiment, the animal is a mouse.

[0082] In one embodiment, the animal is a female. In another embodiment, the animal is a male.

[0083] The present disclosure is further directed towards a genetically modified nonhuman animal or animal cell whose genome comprises an exogenous DTR gene inserted in the endogenous Jchain gene of the non-human animal. In one embodiment, the cell is an embryonic stem cell. In another embodiment, the cell is an embryonic stem cell of mammalian origin. In another embodiment, the cell is an embryonic stem cell of rodent origin. In a further embodiment, the cell is an embryonic stem cell of mouse origin. The present disclosure is further directed to a primary cell culture thereof derived from the non-human mammal or an offspring thereof. As used herein, the term “cell” includes cell lines.

[0084] Joining chain, or Jchain, is a small polypeptide that regulates the multimerization of IgM and IgA and is expressed in antibody-secreting cells (ASCs). The mouse Jchain gene comprises 4 exons. Exon 1 encodes the 5' UTR and the leader peptide, exons 2 and 3 encode the N-terminal half of the protein and exon 4 encodes the C-terminal half of the protein as well as the 3' UTR. Sequences of the mouse Jchain gene and protein are well known in the art (see for example Gene ID: 16069 for a mouse Jchain gene and Accession number NP_690052.2 for a mouse Jchain protein).

[0085] Diphtheria toxin receptor (DTR) is a protein that confers toxin-sensitivity diphtheria toxin. Diphtheria toxin is an exotoxin secreted mainly by Corynebacterium diphtheriae. DTR is the principal protein involved in binding diphtheria toxin to cells, and thediphtheria toxin sensitivity of cells is determined primarily by the presence or absence of DTR. DTR is encoded by the DTR gene.

[0086] In one embodiment, the DTR gene is human or simian DTR (also known as simian HBEGF).

[0087] In one embodiment, the DTR gene comprises or consists of the DTR gene from Chlorocebus sabaeus (NCBI Reference Sequence: XM_008014669.2; SEQ ID NO: 1). In another embodiment, the DTR gene comprises or consists of SEQ ID NO: 1 or a functional fragment thereof, or a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to SEQ ID NO: 1 or a functional fragment thereof.

[0088] In another embodiment, the DTR gene encodes an amino acid sequence that comprises or consists of the amino acid sequence of the DTR protein from Chlorocebus sabaeus (NCBI Reference Sequence: XP_008012860.1 ; SEQ ID NO: 2).

[0089] In another embodiment, the DTR gene comprises or consists of the DTR gene from humans (NCBI Reference Sequence: Ref Seq: NM_001945.3), a functional fragment thereof, or a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to Ref Seq: NM_001945.3 or a functional fragment thereof.

[0090] The term “functional fragment” and "functional variant" as used herein with reference to diphtheria toxin receptor refers to a protein (or a gene that encodes for a protein) that retains the biological activity of diphtheria toxin receptor, namely susceptibility to diphtheria toxin. Functional variants and fragments encompass, for example, gene variants of SEQ ID NO:1 that retain the ability encode for a protein that confers susceptibility to diphtheria toxin and protein variants of SEQ ID NO:2 that retain the ability to confer susceptibility to diphtheria toxin.

[0091] As used here, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide asthe corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences ( / .e., % identity=number of identical overlapping positions / total number of positions multiplied by 100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBI_AST programs of Altschul et al. (1990). BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecule of the present disclosure. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997). Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Altschul etal., 1997). When utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988). Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GOG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0092] As noted above, the mouse Jchain gene comprises 4 exons. In one embodiment, the DTR gene is inserted into an exon of the mouse Jchain gene. In another embodiment, the DTR gene is inserted into exon 4 of the mouse Jchain gene, optionally the 3’-UTR of exon 4 of the Jchain gene. In another embodiment, an IRES-diphtheria toxin receptor (DTR) cassette is inserted into the 3’-UTR of exon 4 of the Jchain gene.

[0093] In one embodiment, the genetically modified animal expresses Jchain protein at a level of at least 50%, 60%, 70%, 80%, 90% or 95% of the level that a wildtype animal expresses Jchain protein.

[0094] In another embodiment, the genetically modified animal expresses DTR in antibody-secreting cells (ASCs).II. Methods of making genetically modified non-human animals or cells

[0095] Another aspect of the present disclosure is a method of making a genetically modified non-human animal or animal cell comprising modifying a genome of a non-human animal or animal cell to comprise a diphtheria toxin receptor (DTR), wherein the DTR gene is inserted in the Jchain gene of the non-human animal, and generating a non-human animal or animal cell comprising the modified genome.

[0096] In one embodiment, modifying comprises introducing a nucleic acid molecule comprising a DTR nucleic acid sequence into the genome of a non-human animal embryonic stem (ES) cell, obtaining an ES cell in which the DTR nucleic acid sequence has been inserted into an exon of the endogenous Jchain gene, and generating an animal from the obtained non-human animal ES cell.

[0097] It is known in the art that genetically modified animals can be made by several techniques including but not limited to nonhomologous end-joining (NHEJ), homologous recombination (HR), zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. In some embodiments, homologous recombination is used. In some embodiments, CRISPR-Cas9 genome editing is used to generate genetically modified animals. Many of these genome editing techniques are known in the art, and is described, e.g., in Yin et al., “Delivery technologies for genome editing,” Nature Reviews Drug Discovery 16.6 (2017): 387-399, which is incorporated by reference in its entirety. Many other methods are also provided and can be used in genome editing, e.g., micro- injecting a genetically modified nucleus into an enucleated oocyte, and fusing an enucleated oocyte with another genetically modified cell.

[0098] Thus, in some embodiments, the disclosure provides inserting in at least one cell of the animal, at an endogenous Jchain gene locus, a sequence encoding DTR. In someembodiments, the modification occurs in a germ cell, a somatic cell, a blastocyst, or a fibroblast, etc. The nucleus of a somatic cell or the fibroblast can be inserted into an enucleated oocyte.

[0099] In other embodiment, a method of making a genetically modified mouse is provide comprising (a) inserting an exogenous DTR gene into the Jchain locus of a mouse embryonic stem cell (ESC) line, (b) microinjecting the resulting ESC clones containing the insertion into blastocysts and transferring them into pseudo-pregnant females, (c) mating the resulting chimeras with wildtype mice and identifying germline mice as heterozygous for expression of the DTR gene in the Jchain locus.III. Methods of use

[0100] Also provided herein are methods of depleting ASCs in a non-human animal comprising administering diphtheria toxin to a non-human genetically modified animal as described herein.

[0101] In one embodiment, a single dose of diphtheria toxin is administered. In another embodiment, a single intraperitoneal dose of 150-200ng diphtheria toxin is administered, optionally 200 ng diphtheria toxin or about 200 ng diphtheria toxin.

[0102] In one embodiment, the ASCs are IgM, IgG and / or IgA ASCs.

[0103] In another embodiment, the diphtheria toxin is administered after the genetically modified animal is vaccinated. In a further embodiment, the diphtheria toxin is administered after the genetically modified animal is infected with an agent of interest.

[0104] Further provided herein is a use of diphtheria toxin for depleting ASCs, wherein the diphtheria toxin is for use in a non-human genetically modified animal as described herein. In one embodiment, the genetically modified animal is vaccinated. In another embodiment, the genetically modified animal is infected with an agent of interest.

[0105] In one embodiment, a genetically modified animal as described herein is used to study ASC differentiation kinetics, for example by administering at least one dose of DT to the animal and assaying ASC numbers at different time points. Also provided is a method of studying ASC differentiation kinetics, comprising administering at least one dose of DT to agenetically modified animal as described herein and assaying ASC numbers at different time points.

[0106] In another embodiment, a genetically modified animal as described herein is used to study ASC expression in different tissues, for example administering at least one dose of DT to the animal and assaying ASC numbers in the different tissues. Also provided is a method of studying ASC expression in different tissues, comprising administering at least one dose of DT to a genetically modified animal as described herein and assaying ASC numbers in the different tissues. Examples of tissues where ASCs are expressed can include, but are not limited to bone marrow, spleen and thymus.

[0107] In another embodiment, ASC generation and survival following vaccination can be tested in the presence or absence (i.e., DT depletion of pre-existing ASCs) of competition for survival niches. In another embodiment, ASC generation and survival following pathogenic infection can be tested in the presence or absence (i.e., DT depletion of preexisting ASCs) of competition for survival niches. This can also be applied using both young and old animals thus studying the aging immune response which has become even more critical given the recent SARS-CoV-2 pandemic (ie. , Covid-19).

[0108] Genetic, molecular and behavioral analyses for the genetically modified animals as described above can performed. The present disclosure also relates to the progeny produced by the genetically modified animals provided by the present disclosure mated with the same or other genotypes.IV. Nucleic Acids

[0109] Also provided herein are nucleic acid molecules for use in the methods described herein.Targeting Construct

[0110] Provided herein are targeting constructs for use in inserting DTR into the mouse Jchain locus. In one embodiment, the targeting construct comprises an IRES sequences upstream of a DTR nucleic acid sequence. In another embodiment, the targeting construct comprises SEQ ID NO: 17 or SEQ NO: 18 or a variant thereof having at least 75, 80, 85, 90, 95 or 99% sequence identity to SEQ ID NO: 17 or SEQ NO: 18.Primers

[0111] Further provided herein are primers for use in methods described herein. In one embodiment, the primer is for use in confirming the appropriate insertion of the DTR into the mouse Jchain. In one embodiment, the primer comprises or consists of any one of SEQ ID NO: 3-12 or a variant thereof having at least 75, 80, 85, 90, 95 or 99% sequence identity to of SEQ ID NO: 3-12.Examples

[0112] The following non-limiting Examples are illustrative of the present disclosure:Example 1

[0113] A mouse model was generated in which the diphtheria toxin receptor (DTR) was knocked into the endogenous Jchain locus without disrupting Jchain expression (FIG. 1). This led to high levels of DTR expression by ASCs in multiple tissues (FIG. 2 anf FIG. 3). To determine the functionality of this model, studies were performed that acutely depleted ASCs following a single treatment of diphtheria toxin (DT) (FIG. 4). This resulted in the targeted depletion of ASCs in the bone marrow, spleen and thymus of Jchain-DTR expressing mice. Notably, DT had no effect on ASC populations from wildtype animals. In summary, the Jchain-DTR mouse is a reliable and convenient model to acutely deplete ASCs which has the potential to be applied in numerous research settings.Generation of CTR cDNA co-expression knockin mouse

[0114] The mouse Jchain gene was modified by inserting (“knocking in”) an IRES- diphtheria toxin receptor (DTR) cassette into the 3’-UTR of exon 4 of the Jchain gene. Germline-confirmed F1 heterozygous mice were obtained.Identification of Germline Neo Deleted Mice

[0115] Targeted iTL BF1 (C57BL / 6 FLP) embryonic stem cells were microinjected into Balb / c blastocysts. Resulting chimeras with a high percentage black coat color were mated to C57BL / 6N WT mice to generate Germline Neo Deleted mice. Tail DNA was analyzed as described below from pups with black coat color.Screening for Introduced Knockin Cassette

[0116] A PCR was performed to detect presence of the knockin IRES cassette (FIG. 5). SQ1 is upstream of the knockin on the long homology arm and SQ3 is downstream inside the knockin DTR sequence. SQ1 / SQ3 amplifies a PCR product 1.03 Kb in size.

[0117] The following is sequencing of representative mouse #548 using primer SQ1. The sequence shows retention of the knockin cassette. Genomic sequence is in plain text, and the knockin IRES cassette is in bold (TTACTGGCCAAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTC CACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTG ACAAGCATTCCTAGGGGTCTTTCCCCTCTCNCCAAAGGAATGCAAGGTCTGTTGAATG TCATGA; SEQ ID NO: 18).Query: Sequencing data from PCR products ( SEQ ID NO : 13 ) Sbj ct : Respective targeted all el e s equence ( SEQ ID NO : 14 )Screening for FLP Transgene

[0118] Primer set newFLPI and newFLP2 was used to screen mice for the FLP transgene (FIG. 6). The amplified product for primer set newFLPI and newFLP2 is 330bp. Also an internal control PCR (WTF / WTR) with a product size of -620 bp was used to indicate the PCR reaction was working properly.Confirmation of Neo Deletion

[0119] Primer set PNDEL1 and PNDEL2 was used to screen mice for the deletion of the Neo cassette (FIG. 7). PNDEL1 is upstream of the remaining Neo cassette inside the knockin DTR sequence and PNDEL2 is downstream inside exon 4. PNDEL1 / PNDEL2 amplifies a PCR product 363 bp in size when the Neo cassette is removed.

[0120] Below is sequencing of representative mouse #548 using primer PNDEL1. The sequence shows the deletion of the Neo cassette with the exception of one FRT site. DTR sequence is in italics text, the remaining Neo cassette is in bold text with the FRT site underlined, and genomic sequence is in plain text.Confirmation of Short Homology Arm Integration

[0121] Tail DNA samples from positive mice were amplified with primers NEOGT and A2 (FIG. 8). NEOGT is located inside the remaining Neo sequence and A2 is located downstream of the short homology arm, outside the region used to create the targeting construct. NEOGT I A2 amplifies a fragment of 2.41 kb in length.Germline Neo Deleted Mouse Information

[0122] The following heterozygous mice was confirmed for Germline Neo Deletion.Sequence of JIRE Knockin Allele - Neo Deleted1. Outside 5' Arm (12675 bp) : Plain text (e.g., ATCG)2. Outside 3' Arm (14232 bp) : Plain text (e.g., ATCG)3. 5' Arm (6013 bp) : Bold and italic text (e.g., ATCG)4. IRES (603 bp) : large text, (e.g., ATCG)5. DTR(627 bp) : grey text, (e.g., ATCG)6. Remaining Neo Cassette Sequence (83 bp) : Bold text(e.g. , ATCG)7. 3' Arm (2081 bp) : Underlined text (e.g., ATCG)8. FRT : Bold underlined (e.g. , ATCG)9. Exon (mouse) : Highlighted in grey (e.g., ATCG)10. Oligo (forward) : Highlighted in dark grey italic (e.g. , ATCG)SQl : 5'- GTC AAG TAT TCC TTG CTG TGC AGA TGA TTA GG -3' (SEQ ID NO: 3)PNDEL1 : 5'- GGT TAC CAT GGA GAG AGG TGT -3' (SEQ ID NO: 4)NEOGT : 5'- GTC CGT GTC GCG AAG TTC CTA TAC TTT C -3' (SEQ ID NO: 5)11. Oligo (reverse) : Highlighted in dark grey underlined (e.g. , ATCG)SQ3: 5'- AAG AAC TGC AGC CAG AAG GAG -3' (SEQ ID NO: 6)PNDEL2: 5'- ACT TCT GGG TGC AAA TGG AGA -3' (SEQ ID NO: 7)A2: 5'- AAG CAC AGC TCT TCA TGT CAG AGA CGG -3' (SEQ ID NO: 8)GGTTTGAGCCTCAGGATTTACTTTGTGGAAGGAGAGAGCAGGACTCCTGGAAGTTATCTTCTCTCTCAC ACACTCACACAGAATAAGAAGGAGTCTTTTTTTTTTTTTTAAATAAAAAATTGTCTACTTACTGTAAAT TTTTTCTTTCAAAGTACTTTTGTTTACCCTGCTTGCCATATTAGAGCATTCCTTCATACTATGGTTGTT TGT TAC C TAGT AT AGT GT T CAAGT ACAC AT AT AAAT T AGC CAT GAAT GT AT T AC CT AAGT GT T T ACT T T T AGGT CT GAGT AAAT AAT AT TCT GAT AT CGT GACAGAAAAT T T ACT T CAT GT T GAT AAT GAAT T GAACT AGGTAACCAAATATACAATACAACATCTATAAACATCCAAATTTATACTTATATAGAATGTTTTAGCTT TCTACAGAACTGTTTTATACAAGGATTTATATGTTGAAATAAATGAAGTGTAGAGTCTCTGGACTCCTTTTGGCATCTGCAATTTATTTCTTTGAATGGTCATGGTTTCTTTGAACTCCCAATTTGTCAATGACATTA CAGTCTTCACCTAGACTGTATTCGTCCCTCTTATGCCTGATTTCCACTTATTTTTTTCTATTAGCTTGT ATCTCACTTCTGCTGGGAGGCCTTCTATAACATGAATTATTTCCCCAGCCTACTAATCTCTGCTTCACC ATCATGAAAATATCACAATTATCCACTAGGTTGTATTTGAGTCTGATCAAAGGTTATAATTTATTCCCT TCCAGCCTCACCATGTATAATAAATGCCAATGTATGTTTAACGAAGGAAACTGAACACAAAGCCATTTG CTTTAAAGACCAAGAAAGAAGCTTTGGCCTTACTGCCAGCACTCTTTTAGTTTGTTTTAAAGAGAAATT AT T T T AAAT GAAAAT AT GTACTTGCTC C AAT T AT AC AAGAAAAT T AT GAC C C C C AAAT T AT T C C AG C AC TCTTATTGCTGCTATAATAATTATCACAAACTTCATGATGTAATGATGCAGCCAGCAGAGATATGAACA ACACAGGGACCTCGAAAGGACAGGAATAGACAGACACAGGACTATGGGTTCACTTCAGGAGTGGATAAC TTTTTTTATTAGATATTTTCTTTACTTACATTTCAAATGCTATCCTGAAATTCCCCTATACCCTCCCTC CGCCCTGCTCCCCTACCCACCCACTCCCACTTCTTGGCCCTGGCATTCCCCTGTACTGAGACATATAAA ATTTGCAAGACCAAGGGGCCTCTCTTCCCAATGATGGCTGACTAGGCCATCTTCTGCTACATATGTAGC TAGAGACACGAGCTCTGGGGATACTGGTTAGATCATATTGTTGTTCCACCTATAGGGTTGTAGACCCCT TCAGCTCCTTGGGTACTTTCTCTAGTTCCTCCATTGGGGGCCCTGTGTTCCATCCTATAGATGACTGTG AGCATCCACTTCTGTATTTGCCAGGCACTGGCAAAGCCTCACAGGAGACAGCTATATCAGGGTCCTTTC AGCAAAATCTTGCTGACATATCCAATAGTGTTTGGGTTTGGTGGCTGATTATGGGATGGATCCCCTGGT GGGGTAGTCTCTGGATGGTCCAGGAATGGATAACTTTTATTTTCTCAAATGCTCTTTGTATATTGTTTA GCTAGCTACTGGAGATGGCATTGAAAAACAGAGCTTGGACAGTCTTTATGATTCCTTATCCAAGGCCCC ACTTCTGCACTTTCTGCCCTTGTGATCTTGTGTTCCCTCAGAAGACCTTCAACCCAGCCTTAGTGCCTG AAGTTTTCCAGCCACATGGCTGTTGTGGTGAATTGTTAATCCTCTCTCAGCTTCTGGAAGGTGATCACC CTTCTTGCCATCCACTCAGAGTTCTCTGGATTTGTGAATGAAACACACACACACACACACACACACACA CACACACACACACACTCACACACAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGCCTC ACTAGCTCAGTGAGTGATTGGCCACTTCCTGAACCTCCCTGAGGCTAGCACACACTCCCTTCCAGTATT CCTGAGTTAATACTTCTTAAATCTATATTTTATCTTTGCTGCCCTGTTTCTGGTCAGCCCTCCTATGGG GTCGCTTTCCCTTTTACCTACATTTCAGTCGTCTCACCCTCCTCTCCTTCACCATGTGGTCAGTCCTAT ACTCCTCTCATGGCAGAATCCCTACTTCCTTCCTTCGTCCCTTGTCTGGGAAATCTAAAAGTCCTGCCT CCGTCTCTCTGCCCAACCATTAGCCATTGGCAATTTTATTTCCCAGTCAGAACCAACTGGGGGCTCTCT TTCTTTAGCCTTCATTTGGAACATTGCAAACAGGTTTTTTTGGGTAAAACAATTAGCATGATTACACAA GCCGATATATCTTCCCCCTTTTCCCTTTCCGATATACATTGAACAATAAAGGTTTTTCTGGTTACTGCG TGTACTGGCTAGTTTTGTGTCAACTTGATACAAGCTGGAGTTCTCACAGAGAAAGGAGCTTCAGTTGAG GAAGTGCCTCCATGAGATCCAGCTGTAAGGCATTTTCTCAATTAGTGATCAAGGGGAAAGGGCCCCTTG TGGGTGGGGCCATCCCTGGGCTGGTAGTCTTGTTTCTATAAGAGAGCAGGCTGAGCAAGCCAGGGGAAG CAAGCCAGTAACTAACATCCCTTCACAGCCTCTGCATCAGCTCCTGCTTCCTGACCTGCTTGAGTTCCA GTTCTGACATCCTTTGGCGATCAATAGCAATGTGGAAAGTATAAGCTGAATAAACCCTTTCCTCCCCAT CTTGCTTCTTGGTCATGATGTTTGTGCAGGAATAGAAACCCTGACTAAGACACTGCCTCAAGGATATGC T GAT T T G GGAGAAAT GT T T T T T CAT T T T T AGAAAAGGT GAT T AGT GT CT AT ACAT C T T C CAGAGT AAT A T GGAACAGAT T T GAT AGAGGAGAC C C C C CAGAGAACT AGAT T C CAGAGAAT CAAAC AAAAAAAAAGT TA TCTTGATGATACTAAAATTTTGTTTTGAGATTTGTATATTACAGAATATACAGCCTTGGTGAGTTTCAT CTTGTGGGTGCAATAGCATAGATGTATGTGTTTGTGGAGGGACAACTCGAGTCTCATCCTCAGGAGTCC ACTTCCTTTGAGATGGGTCTCTCATTCATTCAGAGCTCACCAACTAGGACACACTGGCCCGTGAAAACC ACGGATCCTCCTTCCTGTGCCTCCCTCCTCAGCATTGAGATTCTGAGCACTGCCATGCCAGCACACCTG ACATTTACATGGGTTCTAAACATCAAATTCAGCTCCTTGTGCTTACAGGGCAAGTGCTTTTACTGACTG AGCCAGCCATAGCCCTGCACTTGATATTTTTAAATAACTGATTGGTCATTTTTCATGTATTATTCTTTT TCCTTGTTCT CAAGAAAAAT GT AT T T AAC GT GGT AAT GAT G GCAAAT ACAAACACT GAT AAAAT AAT TA GAACAAGGAAAAATGTATCTTCTGACCTATATTCCCTCTCTCACTTTCTTTTCTCTCCTTCCTTTCTTTCTTTCTTCTTTTTTATTTGGTTTTTCGAGACAGGGTTTCTCTGTATAGCCCTGGCTGTCCAGGAACTCACTTTGTAGACCAGGCTGGCCTCGAACTCAGAAATCCACCTGCCTCTGCCTCCCGAGGGCTGGGATTAAAGGCGTGTGCCACCACGCCTGGCTCCTCTCTCACTTTTTTACTCTCTGTAGAGCAGTGGTTCTTTAATATAGCCTAATGCTGCAACCTTTAGTTCCTCATGTTGTGGTGACCCTCAACCATAAAATTATTCTTATTGCTACTTCATAAGTGGAATTTTGCTACTGTTATGAATTGTAATGTAAATATCTTTTATGCAGGATATCTATATGTGACTACTGGACATGACCTACAGGTTGAGAACTGCTGTTCTAGGTCTCACACTAACTGATACTAGCCTGGGATTTACTATGTAGCCCAGGCTGTGTTCAAACTCCTGTCGATGTTCCTGCTTCAGTCTTCTGAGCACCAGAAT TATAGAAAT GAGT CT T GCT CT TTAAT CT AACAAAT CTACT GTATAT GTATAT GCAT GT GCTT GTAT GGGCT CATATGT GT GT GT GT GTACAT GT GTAT GCAT GT GT GT GT GGT GTATAT GT GCAT GT GT GT GTGGAATGTGTGCATATACACAAATGTGTGCTTATGTGTGTATGTGCTTATGTGTGTGTTGGTGGTGCA CACTCATTGAACTATATCCCCAGCCCTGTGGTGCCCATTTGGTTTCTCTTCATATTCTTTTTTTAAGTG CCATTTGAAACTGCTCTTTAAAAACCCCACTGTGCTTCAAGGAAGCATTCATTTAGTGTAAGAGAGAAA TTATATACATATGAAGAAAATGACTTTAAAAAGGTGAAGGAATGTGTTAATTTATATAAGGTAATGTGG GGACACAAGTGGAGTTTATTGCAATCTATTTTAATAGTAAAGGGTGACTTGAATTAAGTTGTGACTTGC TAAGATTAAATTAAAATACTTTATGATTTTTGATAAGGATTGTGTGGTAAACGGTTAAATTTTTGGTAA GGAAAT GACAGAATAAAAGAAT T GTAAAACAGGAAACAGCAAATAGTATAT CACAGGAAAGGAAAGACT CCATATTTGCCTGTAGCAGCAGCCTCCTCCCCAAATAGATGATACATTTCCTTAAAGCACAGCTAGACC AGAGTACTGGGGCTGACGGACCAAAAACGGGGTTTGAAGGATTCTCTTGGGACCATGTTTGTTACCATG TCAACAGAAAAATGACCATGAACTCTAAGCTTATTTTTTTTTTCCCAATGAAGTTTGGAAGTAGACCAC TTGTCGATTAGGACCTGTGGGTAATGGTTAGCTTTGGGTTCTGCCAGTCAGCCAACAGGCAGATCCCAA TAGCAATTCTATGAAAAGCCACTGCAGGTGCAAGTTTATTTAAAAACAGACCACAGCCCAGGTGCAATT CTCCTTATAAACATTTTAACCACAAGCCTGCCCTAAGAGTCTGTGTGGAAAGGTATTTTGAAAAAAAAA TGAAGTTTAGCTTTTTTCTTGTCTTAAATTTTTGATTTTTAGAAACATTTTGTAATAAGGTTTCTCCCT ATGTTTTGAGTTCTTTTTAGATTTAAGATTTTGCTGTCTGATACCCAAATAGAAATTTGACAAGTACTG TGATTGGCTAAATACCAATAATGAAATTTCTGTATGCAAAAGCCAAAATATTTAGGGCTAAAATGCTAA T T AGAAGAGAT T T CT C CAT ACAGAT GT G GAAAAAAT GAGT T T AT AAGAT T T T T GGGAC GT T GG GAGGT A AAATGAACTGAAAGTTAGTCCCTGGTTCCTTCCACTTTTGTCTTTAGAATCACTGAGACTGATTCTAAA ATTCAAATTAGAGTCTAGTGTGTGCACCATACACCATGATTCCAGTAATTCAGAGTTGGAGGCAAGGAA ATCAGGAGTTCAGGGCTAGCCTCATCTCCATTGCATGTTTGAGGCTATCCTGGGTTACATGAAACCCTG T GC CAAC AAAGGAGGAAACAAACAAAAAT C CAAAAT AGAT TAT AT T T T T T AT GTAT TTCTTTTTGTTTT GTGATTTTCTTTTTTTGAGACAGAATATCATGTGACCTTAGTTACTATGTAGCTGAAAAATGACCTCAA ACTTGGTATCTTTGTACCTCTATCTCACAAAGTGTTAGGATTATAGGCATGAGCCACTGTGCCAAGAAA AACATGTAAAATTTAAATAGGAAAAAAATTTCTCTTGGACTAGTGAGATGGCTTAATGTGTAGAGAAAC TTATGGCAGAAGCCTAACCTCCAGGTTAGTGGAAGGACAGTACTGACCCCCAAATGTTGTCTTCTAACC TCCACATATGCACTGGGCATACATGGCTGCACTCACACAATTATACACACACACAGTAATAATATTAAT AATAGTAAAGAAAATGATTGTTACACATTTCTCTTATAAGGAAGCATATAATGTCATTTACATTTTCCT CCTCACTCTGCTGCATGTAGGGGGTTTTCTTCCTTGTTCCATGACCTGTGCAGACATGTCTTCCCCATC TAATTCCATTCACTTGATGTTTCTTTTATTGCAGTAGTATTTTTTTATTTTGTTCCTGTGATAGGCAGA AAAATGACCCCTTCACAATGCCCATGCCTTGTGCAGGACAGATGATGTAATAGTTAAGAGTACTTGTTC TTGGAATTTGTGACTCTAGCTGTATATGTAGCAGAGGGTGGCCTAGTCGGTCATCAATGGGAGGAGAGG CCCTTGGTCCTGTGAAGGCTCTATGTGCCAGTATAGGGGAATGCCAAGACTATGAGTGGGAGCAGGTAG GCTGGGAAGCAGGGGTTGGGGGGAGGGGATAGGGGATTTTCAGAGGGGAAACTAGGAAATGGGGTAACA GAAATGCAAATAAAGAAAATATCTAATAATAATAACAAAAAAGAGTACTTGCTCTTGCAGGAAAGCCAG GCTCTATTTCCAACAACATCAAGTTATGGTTGCACCATTCATAACTCTGGTACCAGGGCATTCAGTGTC CGCCCCTGGTCCCAGTTTCTGATTTTGATGTTAAAGGGGGGGATGTTCTCAGGTTCTAGAATCTCAGAA AACTTGAAGCCAGCAGGGCAATGGGGAATAGGTTTTAACAAAAGCAAGAGATAAATTCTGCCAACAGTT T GAAT GAAT TT GAAAGT GGCTT AT CT CT TAGT GT GTT GAGATAAGAGT CT GGGGACAAGGAAT ATAGCT TGGTGACAGACTGCTGTCAAGCACATGGGCCTCCAGTACACTGCCAGGGCAAAAATAACCAAGAATTCC CAAATCGACAGCTTAGGTTGACAGACACTTCAGACTAAGATGCAAACTTGGAGTGACCTGATTAGCCTG GCCTACCTATGGGTGTAACACAACTCCAGAAATCATCAATGGTGGTATTTGAAATCACTCTGTACGGTC ATTTGTTATGAAGCTTCATCTTGTTTGTTCATGTTTCTGCCTGGATGTGCCTCCTCAAAGCCTTTCCCA AGAATACTCTCTGATGGAGGTGACCCCAGCCCTGCTTCACATGCTCGTCATATTATTCTTGTGTGGATT TCTTTTTGAGCGTGTATCACAATGTGTGAGTTTTTATTTAGCTTTCTTTTTCCTGCTAGTCTTCCTCAT CAGAATATAATGTCTGGAAGACAGGGCCTTTTAGAACGTAGAAAACAACACAGCAAGGGTCATGCTGGG GAGGAT ATAT TAG CAGT AAGAACAAAT G GT T T AGC T AC GT T T TAT C GT AG GGAAAT C CAC CAT C CT C C C TTTTCAGACAGCATCTGTCTTTGTATCCTCTGCTGGTTTTGAATTTATAGCAGTCTTCCTGTATCAGCC ACATGAGTGCTGAAATTGCAGGAGTGAGCTACTATGCCCAGCTAAAAAAACTCTGTCCTCTATTAGTAA CCAGGTACATCACTGATGGTGGTAACCTGGGACATTTGGGAAGCTTAGTTTGTTTAATGTTGCACTGAT ATTGAATATATTTCACAAGTGTTTTATACAAACACCTGTTTCCACTTCTGAATGCTTTGTGTTTATGAA TTCACTGTTCTCTGCTTTGAAGATTTTACCTCTAGTTCTAATAGAATCTTCCCTTAGTTTACTTCTTTG AGTTGATCCAACAACTCTTATCTTCCCCTAATCCTAACCCTTTCTGCTCTGCCTTTAGTGTCAAAGTCATAAAAGTTAGAGAAAGAGTTATATGGAGATATGGTAAATGCTGTGTTTCTGACGGGTCACTATCATAGCTACATGGTAAGCTGGGTTTTGGGGAGAGAACTCTCCATGTCAGTTTCCTCCTTATATATTGTTTTTGTGGGAGTGCCAGTAAGTTGTGGTTGGCTGAGTGTGTTATACAGCACCTCAAGCACATGGGTTAGTCTTCTTCTAAAATTATTAAATTTCCTTTTTAGAATTGATGTGTTTAGAGTCTGTTTACAAGAATGCTAAGATAAATCTGAAGGCAGATACATTTTTAGAGAATTTGAAGGATGACTGGTCTCTCTTACCTCCTGTATCCTTTATGATAATACCAAGGGAACTATCAAATATATTAAGGAGTTCACATATGTATTGTACCTTTTGTAGCATTTTTGGGAATACTCAGAAGTCATTTTAATAGGGATTTATTTTTTATGTATATAGGTGTTTGTCTGCATGCATGTGTATGTACTATGTGTGTGCTGTGTATCCAGGGAAGCCAGGAGAGGGGATCAAACTCCCTGTACTAGAGTTATAGATGGTTGTCAGCTGCCACATGGATGTTGAGAACTGAACGCAGGTCCTCTGCAAGAACAGCCAGTACTCTTAATTATTGAGCTTTCTCTCCAGTCCCAGAAGTTTTTTTTTAAAGTCGAAAAGATTATTAGATTATTCGAAATTTATAGATTGACATTTAGTTCAATATTTAATGTGGACTTTGTTTTTTCTATTTGTTGGGTGATTTGGGAAATGTTTATTTATTATTAAAACTTGTTTTTCCACTTCTATTTCAAGGTTAAATGGGAAAT AT GAAGC AT T ACACT AAGT GT AGAGT GT AAT C CAT T AT AAAGAAT AT C AAGAGAT TTCTGCTC CAT CTTAGACATGCAGCCAAGAAGGCTATTAACATATCACCTCTTTAAAGTGTCCAAGAGGCAGAATGCCAGGCTGCTCTCAAAGATTCCCTTATATGAGAGGAGGCTTTTACAGGGAGAAAAGTCACTGAAGACAGAGGATAAAGGAGAGATTGAGCATATTGCCAAGTACTCCTTGTTCTTTTTTGGATGAAAAGGGAAATTTCAGTCTGAAGCTGGCCGTGGTTGCTCATGCCTGCAATCCTAGCACTTGGGGCAGAGGCTGGAGTTGAAGCTCATACAGAGCACACAAGTTTTAGTTCAGTCTGAGATGCAGTGTGAGACTGCTCCTCCTCCTTCAACAATAGCAAGAAAAC AACAT T T C AGT CT T T T GGT CAT GT AT CT AT AT GAC CACAT GAT T T GACT T GAAAT GAGT T GATGCACACAAATGTACTTATTAATGGGTTGTTAGGCTGTAGATAGGACTTTAGAGGACTAGCTTAGAGAACTCCCCTGGCACCCGGCACTACTGCTTCACTGAATGTCTTTCTTGCTTGCTTGCTTGCCTGCCTGCCTGCCTGCCTACCTTCCTTCTGTCCTCTTCTTCTCTTCTCTTCTCTTCTCCCCTCCCCTCCCTCCCCTCCCTTCCCCTCCCCCTCCCCTCCCTCCCCTCCCCCTCCCCTCCCCTTCCCCCTCCCCTCCCCTCCCCTTCCCCCTCCCCTCCCCTCCCCTCTACCCTTCCCGCTCCCCTCCCCTTCCCTTCCCCCTCCCTTCCCCTCTTCCCCTTTCTCTCTCTCTCTCCCTCTCTCTCTTTCTTTCAAAAGATTTACTTATTTTATTTATATGAGTACAC TGTAGCTGACTTCAGGCACTCCAGAAGAGGGAGTCAGATCCCATTACAGATGGTTGTGAGCCACCATGT GGTTGCTGGGAACTGAACTCAGGACCTCATGGAAGAGCAGTCAGTGCTCTTAACTGCTGAGCCATCTTT C CAGT C C CT GAAC AT AT T T CT T AAT AACAT AAAT GAAAT T T AAAACT GAT GGT ACAAGT AT CAT GGT T G CACACATTGTGAAATTGAGGGAAATAACTAATGTGATGGTTTGTAAAACATTGATTCAAAAGGATTGTA ATGTTGGTCCGAATTTAGCAAGACAACACTTAACACTTCAAGTGATTCCAGGTAATCACTAGACTACAC AGGCAAACACTGGTCTCAGACCCTGTATTGCCAGCCTAAGATACTCCCTTCCAGTGACCAAAGTACAGT ACAGAATCATATTTAGAGTTACACACATAGTTATAAACAATGGGCACACTAAGCTCCAAGATAATGAAA ACAGGATGGGGGATAAGATTAAGAGATGGGAAGACCAAACCCAGAGCTTTCCTCCCTTCATCTTAGTCA TCACAAATAACATTATCTGCTTTATACAAAAGAATTTGAGAAAACATTGCTTTTTTTGGGGGATATATC CAAATTTCCTGAGGGAGGCACTTGAGGCTTGAGTATATTTTCAAGGAAATAGTGACTTACACTATCCTT TAACCTTTAAGGGGAAAAGGGAGTCAGGTCTCATGTATCCCAGGCTGACCTTTGATTTGCTATGAAGCC AAGGTTGGCCTTGAACCCACGATCTTTTTGCTTCTATCTCTCTAGTGCTCAGCAATTCATCCATTAATT CTCATTCATATTCTCAGTCTGTGTGCTATAGCCTAAGGTGGTCATTTAGAAACTATAGTTGGGGGAAGA AACCTTAGGTGGTTGATATTTGTTTTGATTTCCATAATTACCACAAACCTCCTTTTAATGTGTTTGCTT TTCTGAGAGAGAGAATTGCTCCCATGATTCCAGTCATGCCCTCTCTCCCCATTTCTCCCTCTCTTCTAT CCTTTTATTCTCCTGGAGTCAGGGTTTTGCTACATTGGTAGATTGGCTTTTAACCCATGATCCTTTCAC TTTAGCCTTTTGACTCCTTGGTTACCAGGAGAATGAATTTTCCTTAAACCTGAGTAATTCTTAAGTCCC ATTCAGAGTAGTATTTAAAATGAGAATACACTTTGATGGTGTAGTGACATCATTTCTTCTAGTGCCTGC CACTTTGTTTTTGCTAGACTTTTACTAAAGATTCTGTTTAATTTTTAAAAATTATTTTATGTGCATAGG TTTTTGCCAGTATGTATGTCTGTGTATCATGTACATGTCTGGTGCTTATGGAGGTCACAAGAAGGTGCT GGATCCTCTGGAGCTGGAGTTACAGACAGTTGAAAGCTGCTATATGGATGCTGGGAATTGAACCTGGAA CCTCTGGAAGAGCAGCCAGTGTTCTTAACAGCTGAGTCATCTTTTCAGTCCCTCACGACAGATTCTTTG AATGAATGTAATTATGTGCTAGGGTTTCATATCTCTGTTGCTTCCTACATTTAGGTTGCCTACTTATGA CCCTGAATTATTGTTTATTGCCTCTCAAAAGTCCAGAAGACAGAGAATTAAAGATAAGAATCAAAATAA TACACTTTGGCTATAGTTACACAAAACATAGTTGTCCTGTTTCCAAGACAGAAAATTCTAGGATGTTGG AGATGGGAGTAGGGGTTTCTCTACTCAGAAACTCAGGTTCGTTCGTTCTTTCTTCTTTCTCTCTCTCTC TCTCATTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTTTACATTTGAAAATAATTAAGTGAAAA AGATTGAGACCAATACTCCAGTGAGTTATTTTTCTGTTGAGACAACATGGATATTGTAGTATGTGGCTC TGAAAGGAATCACTAGGCAAAAAAGTAGAAAGTCGGTCTGTCTTATTAGAGTAAAACGTAACATTAGCTAAGGATCTATTAATTTCAAAATCAGCTTATTAAATAACTGAGAGTGGTAAGAATAAGCATATGGATGCT TAAATATGATAATTTGTTAAAGACTACAGAGAAACTGCTATAATATATTCTTAAAAACACAAAAGGCCT TGATTACTTCTGTTTCTTCTTTTCTTCTCTTTTCCGTCAGTCATCATTCCAGCAGTGACAAAGGTCAAA CAT T T GATT T GAG GT CT AAT AT GAGT AT AT GT AGT T GAC GAAGT AGAGAG CT CAT AT T T AAAC AT T CT C AACAGGGCCCTGACTCTGCATTCTTGCTAGTCTTGGGGTTCATACATTGCTGAAGCACAGATGAGCCAT CAAATCCCAGCTTTCTATTAGAGGGAAACACGTAAGTATGAACCATGTGCGTCTTTCCAGTGTAGCATG CAGTTCAAACCTGAAGCTGTGATGACTGCAGCTCACCTGCTCTGGGGTTATTTTAAGAAAGCAGAAGCA GCATCATTTGCACACCTCTTATAGACACACAGTTGGCCGTGGCTTTTTGGCTTCAGTCTTTCAACAGTG AAGACAAGATGAAGACCCACCTGCTTCTCTGGGGAGTCCTGGCCATTTTTGTTAAGGCTGTCCTTGTAA CAGGTAGGTGGTACCTATACAATAACACCAGATAAAAGGGAAAATGTGTGTTTCAGGTCTAAAGACTTA AATCTACTCTTCTACTGGCTAGCTGGACAGCTAGCTACCTTCTAACCTCATTAGGCAGTGATATATATA TATATATATATATATATATATATATGAATGATTCATTGTATTCTCTTTGTTTTTTTTTTTTTTCTTCTG TTTGGCTACTCCAGCCTACAGCATAATAAAATAAATACTTTGGGGGTATCTATAAGTGTGGCTACACTT ATAAATTCCCATGTATAACTTTAAAACCAATAGCCCTCCCTTGAGTCTCTTTAATTACTATTCTAGAGA AGT GAGGTAAT AACT GT GGT AAT TAT GAT AT AGAT T GT AT T T T GAT T T TTATCTCCCCAGAGAAATGGA GTCAGAGAGTGAAAACTGACTCTCAGTTAAAGCTTCAGCGCACACCTATGCATTTATTCATTTTTCTGT ATAGTGACACTGTACATCAAATTGAACTCATAGCTAACAATATTTTCTAAATTGTTTTTTTCAGCAAAA CTTTGCAGACAGTACTTTACACTGATATATACTCTCACCTTTTATAGAATTCTTCTAGGAAATATTAAT ACAGGCCTTGTTTCTCTTTCTTGTTGAATCTGTGTATTGATTGTGATTATTGTTATGAAAAAATATCGA TAGTTTAGGAAAGTTTTGGTGGAGAGTATTAAATCTTACAAAATAAGATAGTCTTGGGAGAAAAGATAC TTCTAATACGATTTGAGGGAAATCTTTTTTTTCAGGAAGCAATGGTAGTTGCGGAAGGACTCTGTTTGC AGTTGGGTTTATAAAGACTGGCTTTCAAGATTTCTTTGATTGGGGAATAGTCTAAATTTCTGCTTTATT TTTCATTTAGGTGTTTTCACTGAATTTTACCATGTGCCCTCACTAAAATAATTAGAAGCATTAGATATA AATCAGAATTCCTTCAGGCTAGCTGCTCTTGAATTTTTCCAACGAAGAAAAAGAAAATTTCTCTTAAAA ATTCTTTGCTCTTTTCGATTAGAAAAGAAAACAAATAGAAAGACTAGGACTTATGAACAATAAAAATTA AAGTTTTTGATCCAGGCATGGTGGTGCACACTTGTAACCTCAGCACTCAGGAGATGGAAGTAGGGGGTG GAGAATTTAGAATCAACCATAGCTGCATAGTGAGTTTCAGGCTATCCTGGACTACATGAGACTCTGTCT GCTATCTTCAGGTAAGAAAGGCAAATTATCTCATCTTAGCACATAATGCACTTAAAAAAAAAAAAAGAC AGACAATACCTCTTTATGTAATGCAGGGTGTCATGGAAGTTCCCAGGTAGTCCAGGCAGGCCTTGAATT TCCCATCCTCCCTCCTCAATTTCCCAAGCGCTGGGATAGCAGGCCTGCACAGTGACACCAAAGACAACA TAGAGTTCTGTTTTCCTTTTTCATGATATAGTTTACCTTTCAAACCCAAGGTGACGACGAAGCGACCAT TCTTGCTGACAACAAATGCATGTGTACCCGAGTTACCTCTAGGATCATCCCTTCCACCGAGGATCCTAA TGAGGACATTGTGGAGAGAAATATCCGAATTGTGTATGTGACATTCACCTTGCTTTTCTTTTCTCTGTT ATAAGAAGGTACTTTGTGTTAGCAAGCTGTCTTTTCTTTTTAAAGGGAATCATGCAAGGTTTTCTGAAA CAGATCCCTGTAGTAGGGAGGGGCCTTGACAGGGGTTGCCTTGTCTAAATGTAGTGTCTTAACCCACTT TTGATGTGTAGCATCTTTAATAAGATTGAGTCATGAGTTAGATGAAATTGAGTTTTGATAACAAGAAGA AATGAGTTTTGATTTTACAGAAGTCGAGGGCAGAAACAAAGGGAACCGGAGGTTCCTACTTAGTCACCA ACATGCCCGTTTATAAGCAGCGATTTCAGTTTGAATCAGAATTTTCAGTTATTCTTTATCAGCTTAAGC TGCAACTTCAGATAACAGTAAACACTCAACTTTCATTCCACAGTTATTGACTCTTGAGTATTATACATT GTGTTCCTAAAAGACTAATTAAAATTGATTGAATTGACTTTTCGCTCATAAATTGGAGTCACCTATTCA TTTTATTTCTCATATAGCCCAGACTTATATACTGGTAGTGCTTAACTAATAGCAAGAATACTGATAATA CTTGAGAAATACAATTTCTACTTGAGCCAATCAGTGACAAGTTTTTCTTGTTAAATACTCCCTCCTTAA TAGTTTTCTCCAATCAAATACTTCTTGCAGGAAGAGAACATAGCAGTCAATTCCAGCTATCAATAGGAA TATTATGATGTTTAAAAAATTGTTTCAAGATTTAGCTGCATCATTTTGCAATTGTGCACTCTTAACAAA TATCAAATTATTTGCCCACTCGAAATCTAAAATTTCTCATCTTTAAATAGAAGAAAAAACAAGCCTACT TTATAGGATTATTAAAAAAAATGAGAATATCTATTGTTTGTAAAGGTTGTGTATACCAGTCGCATATTT TAACAATCCTGTTATTTACTTCACTATGTGCTTGCTAATCTTATACTGCGGTCCTGAGAAAATGCTGTG GAGCTCGCTTTCCGTATTGTTGTCAGTATGCATTACCTCCAGAGTGCCTTACCTCCCTACCAAGCTTAG CGTAACTCTCCATCGTTCCTGCTATTTCATTCCTATTGAACAAAATGAAACAGACACCAAGCTCCAGGA GTCCTGGAGTGTGTCTTTATTTCTCTACCTTTGCAGTGGGTTTTACCTTTTCTGTTTTTACATCTACTG GATCCTCTGTTTAAGAATTCTTTCTGTTACCCCTTTAAAGCTTGGTTAACTCTTCCTTAGTCTGTGTGT CTAAATTTAGCCATTGAGTTCCACGGGAGGTTTTCTGAAACAGATCCTCATAGCAGGGAGGAGTCTTGA AAGTGGTTGTCTTGTTTATTTTTTGACACTTTAGCCTTTTTTTTTTGGCCAAAATATTGTAAAAATTAA TTTATTTAATTGGAATTTGGAGTATGATTTAAAATGAATTAGAGGGTCCGAATTTATTATTATTATTCTTTAATTTTTTTTTACAGTCCAGTTGTGAGTCCCCTCCTGGTCCACCCTCTGTTCCCCATCCCATTCCTT CCCCACAGCTCCCCTTCCCACTTCCTGGGGCCTCATTTCAACTTTTTATTAATTATTTTATTTATTCAC ACCCCAAGTGTTGTTCCTAGAGTTTCCCACCCCTTCTTCCCCCACCCTTTAGCCTCTGAGAGAGTACCC TCCTACCCTTCCCCCCTTCTCCAGGGCTTCAAGTCTCTACAGGATTAGGCACATACTCTACAATTGAGT CCAGACAAGGCAGTTACTCCTCTGATACTTATGTGCCAGGGGCCTTCAACTCCTATAATCCTTCCCTAA CTCTTCCAAAGGGGTCCCTGACCTCAGTCTAATAGTTGGCTCTAAGTATCTGCATCTGTCTCAGTCAGC TGCTGGTAGAGCCTCTCAGAGGACAGCCATGCTAGGCTCCCATCTACAAGCACAACATAGCATCAGTAA TAGTGTCGGGGCTTGTTTAAATGTAGTAGTTTTAATCCACTTTGCCATGTAACTCAACTTAAAGTTGAG TTTTGTTAACAAGCTTGAGGAAATTTTACAGAAGTTGAAGACAAAAGATTTCAGAACTCTTAAAAGTTA TAGATTCTCACAGAGACATGCAATTAGAGGGAATTAGCCCACAGCATTGCACATAAACCATATTGAAGA ACCACTTATGTTTTGATCTCCAGGGGGACAATTGCCTAAATACACATCTAACTCATCTCTTACTTGTAT TATATTTTACTGATGTCAAATAATTTAGTTATGACTTGATGTGTCATGATGTTTCCTGACAGTGTATTG CCTTTGCTGTGTCATTGTGGAACTGTTCCCAACTCCATCGCTTATTGTAATGGTGCAGCCTTTTGAATC TTATTTCTGACCTGTAAAAAAGAGTATATATATATATATATATATATATATATATATATATATATATAT ACCTGACTATATAATAATTTATACAAGAACGGTATTTGGCTTGTAGTAGAATCTTATAAATGTTTATTT CTCTGTCATTCCCATTACTGAAAACCTTTTCTGGTGTTAATGAGATGGCTCAGTGCATAAAGGTGTTTG CCTGACAACTTGCATTTGATCTCCAGGACACTGCATGGTGGAAGGAGAGGACTAACTCCTACAGGTTAC CTCTGACCTCCACACATGCTCAGCGACATGAGCACACTCACCATCAACACATACATATAGTAAATAAAC AAATGGTAAAACACTTATAAAAGAGCCTTTATAATCAAATAAAAGAATAGATACATTTGTTCTTCAAAA ATGTTTTTATTTGGTGAATAGTCAATGTTGAAGATTAGCACTGGGAATACAATATAGATTATATAAAGT AGTCTATGCTGAACTGCGGATGGTGACACACAGGGATAATCTCAGCATTAGGGAGATGGGACAAAAGAA TCAGGAGTGCAAGGCCATCCTCTGCTACCAAATGAGTTTGAAGCCAATCTGAGCTGTATGAAACACCAT CTCAGTAATGAAACCAATCAACCCTAAACCAAACAATCCCTTCAACAGAAACGGACAAAATGAAAACTC AATAAAGTTCACCCCTAAACAAAAATAATAAAAAAATCCTCTTCACCTACAAACCACAAAATAAAACAG GCTCCAGAAGAATTGCATGGATATGGTGGTGCACACAGACATATGTGGAGACAAAACACCTATACACGT AAAATGAATAAAAATAATTCATCATGTATACATCTTAAAAATACAGATATTTTTGTTTGTTCATTCTAT AAGCAATGAGCATCTACACACCACACATGTAGCCGTCTATAGCCTGAGAAACCAACTAGTGTGTAGACA AGAAACTGATATTTAAAGTATAACAAATTATTGTAAAACCCATGTAATGGTTTACAATGAGTAAGGTGA TGATGAGTACTGGTATAATTAAATAGTAAATACTAATCTGTTAAGACCCACAACCAGAGACAACAATCT TTGTACTTACCTGTTTTACTGTTCTCTCTCTCTTTTCAGTGTCCCTTTGAACAACAGGGAGAATATCTC TGATCCCACCTCCCCACTGAGAAGGAACTTTGTATACCATTTGTCAGACGTGTAAGATTTTCCTTCATA CTTACATATGTTGAAGTTAATAACCTTTAATTAACTTTATTAATAATGATATTTCTGAAAATACATTGG GGTGTAAGCATTGACAAATGTCTGAACTTTAGAATACACCAATTCTTAGGCTAATGACTAGATCCAAAA CTTTGTAAAACTAGGTCACAGAAAAATAGTAGTGGTAAGTATATTCCAAAGAAGAAATTATTGTCATTT ATATAGAAGGGGCCAATTTATTTCAACATTTAAATAAGCAGTGTTTAGCTTTTAGTATTCATGTGTTTC TTTGGTGATGATAGAATCCAATGTTAAACAGCTTAAAAATATTCTCTCACTGGGTGTGGTGGCACCTGG CTCTAATCTCAGCACTCAGGAGGCAGAGGTAGATGGATTTTCCAGTTAGAGGCCAGTGTGGTCTAGAGA ATGTTTCCTAGGCCTGCCAGGGCTATGCAGAGAAACTCTGCCTTGAATCACCCTCCCCTCCCCGCAAAA AGACCACACAGAATCTCCCCTCAGGAGTGTGGTGCAGGAGCAGGACTGACGGTTCCAGGCCAGCATGAG CAACATAGTGAGACACTGTTTCCCCTGAGTGTATTTGTAGTACGTGTGTATCTATGTATTAATATAGAT AGATTCCTCTTGTCACTGGAACTGAATTTATTGAACCATGCACAGAGCTAGGGATAGCCTTATGCAGTG GAAGCTGTGGTACAGTATGGGTAAGATGAGCCTCTCATCATATCATTAGTTATAGTGCTGAGTTGCAGT ATTTTTTACATGAAAGATTCAAGAATTTTGTCAAGTATTCCTTGCTGTGCAGATGATTAGGCCTGGAAA GAATCATCATTTATTGTGCGTCAGAGCTGCAAGAGCAAGCAGACTTCAGACAACATTTATAATGACTTG TATATTGAGATAACATTTGCTGAGGGATTTCTAATGCATGCCTATTCTCAGCTGTAAGAAATGCGATCC TGTGGAAGTGGAGCTGGAAGATCAGGTTGTTACTGCCACCCAGAGCAACATCTGCAATGAAGACGATGG TGTTCCTGAGACCTGCTACATGTATGACAGAAACAAGTGCTATACCACTATGGTCCCACTTAGGTATCA TGGTGAGACCAAAATGGTGCAAGCAGCCTTGACCCCCGATTCTTGCTACCCTGACTAGGCCCCTCTCCC TCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGT TATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGAC GAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACC CCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTA TTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGT ACACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGT GGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAGAATTCTGAGCCGCCACCATGAAGCTGCTG CCGTCGGTGGTGCTGAAGCTCCTTCTGGCTGCAGTTCTTTCGGCACTGGTGACTGGCGAGAGCCTGGAGCAGC TTCGGAGAGGGCT AGCTGCTGGAACCAGCAACCCGGACCCTTCCACTGGATCTACGGACCAGCTGCTACGCCTAGGAGGCGG CCGGGACCGGAAAGTCCGTGACTTGCAAGAGGCAGATCTGGACCTTTTGAGAGTCACTTTATCCTCCAA GCCACAAGCACTGGCCACACCAAGCAAGGAGGAGCACGGGAAAAGAAAGAAGAAAGGCAAGGGACTAGG GAAGAAGAGGGACCCATGTCTTCGGAAATACAAGGACTTCTGCATCCACGGAGAATGCAAATATGTGAA GGAGCTCCGGGCTCCCTCCTGCATCTGCCACCCAGGTTACCATGGAGAGAGGTGTCATGGGCTGAGCCT CCCAGTGGAAAATCGCTTATATACCTATGACCATACAACTATCCTGGCTGTGGTGGCCGTGGTGCTGTC CTCTGTCTGTCTGCTGGTCATCGTGGGGCTTCTCATGTTTAGGTACCATAGGAGAGGTGGTTATGATGT GGAAAAC GAAGAGAAAGT GAAGTT GGGCAT GACTAATT CCCACT GACGTACGTTCGTGGGATTGTGTCC GTGTCGCGaAGTTCCTATACTTTCTAGAGAATAGGAACTTCGTTGGTACCGTACGATATC CTT GAAT CA CTCACCATGCAGCTCGTTGTCCTTAGAGGCTCTCCATTTGCACCCAGAAGTTATACTCACTGCTAATGA ATTTGAAACCAGGGTTTTTTTTTTCCCCCTGTGGTATAAAACTAATGTTCCCTTTCAATCATTAGAATA TCAGAATTGCTGTCATTATTATGTAACTCTCAATTCGAAGTAGTAAATCCTCATGTCTGCTCAAGGAGA TATGTTTAAAAGTTCATTTTCCAGTGTTTAAAATTGTAAGCAAGGGGAGAAAGTAAAATTCCAGGAGTA AAAGTCAAGAGGTTAATGAAACCCAACCCTTTCCTTCCTTCCTTTGCTGTGGAGAGCTGGAGCTTTCGC ACATCCTGTACTAGTCTTTCTCTTAACCTCTCACTGTGTAGAGAAAACAGCCAATGAACACAGGAAGTT ACGTATCTTCACTAGAAGTTATAAAGGACCTGTTTTGGAAATATTTACTAATATTTATGAAAGACTTTT GT T GAAAGT GAT ATAAT T T GAT GCACAAAT GAAAAAAAAT G GAT GT T GAT AT AAT AT AT AGAC T T GGCA TTAGATTTCCTTGATAATATTTTTGACAGTGAGATTTTTATCTGAAATTCTTAAGGGGAGTCTTTGATG T CT CAGC CAAAT C TAAT GAAAT T T GT AT T C CAAAAT AT GT AT T CT CT AGT ACAGT T T GAACAAT T AAAT AGAGTGCTAAGCATATACTCGTGTGAAATGACTCGTTGGGAAAAATTTTGTTTGTGTTTAACCTTCTTA TAT GT AAAT T AAATAT AAGT TAT TAT T G GCAAACAGT T T GT AAGT AT GAT ACAACAGT GCAT T CAT CAC TTAGCTTATATGAGTACATATTTGAGAAATCTTTGTTAATTCACAGGTAAATTTAAGTACAAAGGCAGG GTCCATAGCATGTAGCACCTTTCAGAAGCAGGAGATGTATGTACTCTGTGGTAGGGAGCTTGTTTATCA TGTTCAAGGCCTTGAGTTTGATGTCCAGCACTGCAAACAGAAGAAAAGCAAAGCAAAGCAAAGCAAAGC AAAGCAAAGCAAAGCAAAGCAAAGCAAAGCAAAGCAAAGCAAAGCAAAGCAAAGCAAAGCAAAGCAAAC AACCACAAACTTACACAGCTTCATCCAAGTAGAAAGATCTCACTTAAATGGTGGACTTATTTCAAAGTA ATAACTTTTCACATACAAAGAAATGTTATGGTATGTCAATGATGTATTTCTCTAAGGGCACTATGGAAA GTAGCTGCTGATTCAGAGGCGGACATGAAGATACTTCTGATCTGGGAACAGGGGGTGCACTTTCTATGT CTGCGCTGTATGTTGGCAGTGTATATGAGGTTTAGTAGGCAACTCTAATGGTGCAAGTTGAAGTTTATA TTCTTACATGGCTGAATTAATACTAGGCAAGGTAACTAAATGTAAGATAATTTCTTTTAAAAATGAAAT TACAAATAACTATTTCATAGTTTTTTTTAATAAAAATGATGATATATCCTATCTATCTATCTATCTATC TATCTATCTATATCTATATATGGATATGCATTTCTATCTCATTTAGTATGGCAGTCATAGTAGTTAGCA ATAGTTATTATAATAGTAAATTATAGTTATTTTCTGTGAATTTTCTTTTGGCTCCTCTCTGACAAGGCA ACAAACT AG C GGAAAAAAAAAT AAGT GT T GT AGT T T T T AAAT T CAGAC CAT GAAGGACT T AGAAAAGT A GATTGCTAATTTCCTCCCCCAGAGATGCTAATTAAGTAGTTTTCGATGCAGAAGCTGGAGAAATTGCAT CTCTTACAAATTCCCAAATTCTGCTGCTGTGGGGAACGGCTCAACTGTAGTATATGGATTATGGGCCTCTGCTTCCACCTAGTGGTTGGATTGCAAAGTAAAACAAATAGGAGGTTTTTGGGTTTTTTTTTGTTTTGT TTTTTTGTCTCCTTTAACCTTTATAGACTTATGAAGAGTTCTAGCTTCATTTTATGAATAGCATACCTA AAGTTTTTGAAGATGATATTTATTTATTTAGTATGTGTCCCTGAAACAAGGATAGCTCTTGTAGCTTAT AGCACTCTGACTGCCATAACATCTCCACTGCTATGGGCTGTACCCCCAAAACCATGAACCAGCTTCCTT TCTTGAGTTGTTGTGATTCTTTTTTGAGACAGAGTTTTTCTGTGTGAATGAATCTCTGGCTGTTCCCAA ACTTGCTCTATAAACCAGCCTGGCCTTGAACTCACGGGGATCCACCTGCCTTTCCTGGGATTAAAGGCA TGTGCTACCACTGCCCTGCCCGTTGCTGTGATGCGCAATGCTTTCTATCTAATAGGACCTAGAATCACC GGAGACAAATCTCTAGGTAGGTTCCTGCGGAGTTTCTATGTGGATGGCTTCATCCCATGGATTGAAGTA GTATAGACTGAATAAAGAGAGGTAAGCAAGCTCAGCCAGCTTTCCTCTCTCTCTGCCTCCTGAATTCAG ATGCTCTTGATGCCGCGCCGTCTCTGACATGAAGAGCTGTGCTTGCAAAT GGT GAAT CCAATAAACT GC CTTTTTTAGAGCACCAAAAATAACAGATAAAAAAACTCGGTACTGAGTAGCAGGTCTGTGACTGTGATAAATCTGACCACGTGGTTCAGGGGGTTTTGGAACTGACCAGCCAGAGAAATGTGGAATCCTTTAGAACTA CAGAAGT CT T GAC T G CT AT AAAC CAAAC T T AAT AGAT TAT T AT T T T GT CT T AGT T AAGGT T T C CAT TGC TGTGAAGAGACACCATGACAAAGCAACTCTTATAAAGGACACCATTTAATTGGGGCTGCCTTATAGGTT CATAGGT TT AGT C CATTAT CAT CAAT GT GGGAGTAT GT CAT GGCACAGGAGAAGT GGAGAGTT CT GAAT CTTGATCTGACTTCAGCCAGGAGAAAAAACTTACTTTCTGTACTGGACAGAACTTCAAAGCCCACATCC ACAGAGACACACTTCCTCCAGTAAGGCCACACTTCCTATGGGCCAAATGTATTCAGACTGCCAAATCTG GGGAGGGTGGCATTGAGGAAATCAGACTGCCAAGAGAAATGTGAACAGTGTATTCCTCTTTCATGAAAT TTCAGAGGGGAAAACCTCTGTTGAGAACTGGTATAGAGTCTGTTCATGGTCCATTCTAGAATAGAATGT GGCTATATTCTGCTAGTTCCCTGAATATCTGAGTGAGTCTGAGTTAAAAAATAATAGCCTAAGTTGGTG GGGAGAGGAAATTTCAAGACAGCATAGCATTCAGGCTGTGGCTTATTTACTGATTAGTGTTCGTATCCA GGTTCACAGTGAGCAAGAATATCTTGTGGAGGGCTGGAGAGATGGCTCAGTAGTTAAGAGCGCTGACTG CTCTTCCAAAGGTTCTGAGTTCAAATCCTAGCAACTACATAGTGGCTGACAACCATCCATAATGAGATC T GACT C CCTCTTCTGGTGTGTCT GAAGACAACT AC AGT GT AT T T ACAT AT AAT AAT AAAT AAAT C CT T T TTAAAAAGAATATCTTGTGGAACAGAAAGATATGAAATATGTGCATTTTTGGCAAGGAGAAGAGTGTGA GAAAGTTTATATAAAGTTGGTGATGAAGAGTAAAGAAAGCAGCTATAATTGTTAAAGAGTTAGCACCTT ACACTCACACTGGGACAATAGGAAAAGTATCTTTAGAGCATGACCCTGCCCACCAAAGATTCCACCTTG T GAAAT AAAAT T AAT T T GAAAG GAGATAG C T T AAAC C AAGAAT GT AGT T G AAG GACTCTTTTCTT GAAA ATAACTCCCTATGTTCAGCCTCCAAGGTACACGGGCTATTACAACTATACGAGAAGGGAGCCAAACTACATTTTTT TT TTTT TT GAGACAGGGTTT CT CT GT GTAGCCCT GGCT GT CCT GGAACT CACTTT GTAGACCAGGCTGGCCTCGAACTCAGAAATCTGCCTGCCTCTGCCTCCCAAGTGCTGGGATTAAAAGTGTGCGCCA CCACCGCTCGACACCAAACTACATCTTGAGCTGGCATCAGAATATGGCGGCATTGTCCATGTGGATCTG GATTTGCATACAAACCAAAATGCATAAACTTTGCACCAAGGTTTGGAAAGCTGATATGTGGTATGGTGG GATACCCTGCAGGGATGGTCCAAGAAGCCATTTCCTTGAGTCTTACTTAAAAGCTGTGAAGGTGACACT TAAGTAGCAACAGAGACCTCAGGATGTTGAAGATGCCAGAAGCTTGGGGGGGGGGTCCACTAAGGGAAG TTGGATGGGCAGAGTGGGGTCAGCCTGAGAGCTGGTGCATTGTACTGAGTGTGGTAGAGCTGTAGAGTG GTACTCCTGAGCATCATTAAAGCCCGGATGATGCCACCAAAAGCCCAGATGTTAGACTTGTAGGACTTG GAGTTTGCCCTTCTGGGATTTGTTCTTCCCAGAACAACCTCTCCTTGCTGTGCCTCCATTCCTCACTTT TGGAACAGGAATGCTTTTTCCTTGCCATTTTATGTTGGAAGCATTCGACTTACTTTATTGGCACACTTA ATAAATCATTTTGTGGGGCTGGAGAAATGGCTTAGTAGTTAAGAGTGCATTATAACTCTCACAGAGGAT CTGAGCTTTCTTCCCAACACACATGTCAGGTGGCCCACAAGTGCCTGGAACTCTAGCTCCAAGCGATCT GATGTCTCTGGCCTTTTCAGGTACTCCGTAAACACCTATCACACTCACACAAATGCAGACACACATGGT TAAAAATAAATTAATCCATCTACCTGGAAATCTCATGATATCCCTTTTATTTGCAGTTGAATAATATTT CATTTGTGTATATGTGGCATGTTTTCATTACCCATCTATTGAAGGACATTTAGGTTGTTTCTATTTTTA TTTTCTGGCTATAATGAGTAGATTGGCGATGAAACTGGCCAAAGAGTATAATACCTTTGTGTCTTTGCC AAAGAGTGGTATAGCTGGGATATATGGTAGATGTAGTGTTAGTTTAGTTTAGCTCAGCTTAGGCTTTTTTTTTTTT GGGGGGGGGGGCT CT CCATACT GATTT C CAGAGT T GAT GCACCAATTT GCATT CCCACCAATAGTGAATGAGGGTTCCTCTTTCCCTACCATTGTTACCATTTATGATCCATTGTTTTGTTGATCTTAGCC CCTCAGAGTTGTTTTAATTTGCATTTCTAAGGATGATGAGTATTTTTGAGGTATTTCTTAGCTATTTTT ATTTCTTCTGTTGAGAACTCTCAGTTTAGATACCTAGGTCGTTTTTAAACTGGCATCTTAGGGTTTCTC CTGCTGAGACACCATGACCAAAGCAATGTTTGTTTATTTATTTATTTTGGTTTTATGAAACAGGGTTTC TCTGTGTAGCCCTGGCTGTCCTGGAACTCACTATGTAGACCAGGCTGGCCTCAAACTCAGAAATCTGCC TGCTTCTGCCTCCCAAGTGCTGGGATTAAAGGCGTGCGCCACCACTGCTCGGCCCAAAGCAATGTTTAT AAAGGACAATATTTAATTGGGGCTGGCTTACTGGTTTAGAGATTCAGTCCACTATCATCATGGCAGGAA GCAT GGCAGCAT C CAGGT GAAAGAGAT GAGTT CTACAACTT GATAT GAAGGCAGT CAGGAGAAGACT GG CATCCTCAGGCAGCTAGGGTAGCTATAAAGCCCACCCCGATAGTGACACACCTCCTCCAATAAAGCCAC ACCTCATAAGAGTGCCACTCCCTGGGCCAAGCATATTCAAACTACCACAACTGGGCCATTTTTTTTAAA AACAAACTTTTTTTTGGTTGTTGTTGTTGAGTACTTTGTGTGTTGGCTAGTTCTACGTCAACTTGATAC AAGCTCCAGTTATCTGAAAGGAGAGAACCTTTCAGATTGAGAAAAAGCCTCCTTAAGATTAGGCTGTAG GTCTAGAGAGATGGCTCAGTGGTTAAGAGTACTGATTGCTCTTCCAGTGGTCCTGAGCTCAATTTCCTG CACCCCGGTGGCAGCACATAACTGTCTGTAACTCCAGTTTCAGGAGATCTGACACCCTCACAACAAATT ATATAGAAT AAAGTT AAATAAATTAAAAAAATAAGGCT GTAGGGTATTAT CAT AAT TAGT GAT T GAT GG AGGAGGGTCCAACCCATTGTGGGTGGTGCCATTTGTGCACTGGTGGTCCTGGGTTCTATAAGAAGGACT GAGCAAGTCATGAGGAGCAAGCCTATAAATAGCAACCCTCTGTGGCCTCTGAATCAGCTCTTGCCTACA GGTCCCTGTCCTTGAGTTCCTGCCTTCACTGCTTTTGATAATGAACTGTATTTTTTTTTGTGAGTGAAATAAACCTTTTCCTCTTCAAGTTGCTATTGGTCATGGTGTTTCATTATAATAGTAGTAACCTTAGTGAAG ACAAATTGGTACCAGAGGTTGGATATTGATTTGACAGACCCTGATCTTGTAGAATTTTGGGAGGATTGT GGAAGAATTGGAACTTTGGGTAGTAAAGTCATTGAGTTTACCAAACGCTGGAGCTGAAGAATCAGCTGT GGTTAAGAAGAGACTACCATCATTGAGGAGAAATCTTCCAGGAACTGTTTTCTGAGATTAAGCACATGG AAGTTGTGGTGGCAGCTGAACTCGGTACTGTGTAAGAGTCACTGAGATGGTGCTTGTTTTGAAGGTGTT AAGGGGTCATAGAGAACAGCTGAGGCTTGGTGCTGTGTGGAATCCCGAAGACTGTCCAGGGAAGACTAC TGGTAAAAGTGTAGCCCTGTTGAAGCAGGAGGCCCCAGCATTTTGGAGAAAACAGTACTATGGGGTGAC CACCAAGAACAACAGCAGTGGAGGTTTGGAGCCAATGGGAGCCTAGAAAACAAGCTGTGTGTACTGCAG AGGGCAGAGCCAGAGAAGTGATCCAATTCCACTGGAGGAGCCCAGAAGATCATGAGTGAATCCCAGATA CTGGACCTATATGATTTATTATTGTATTTATTGACCTTTGCTTATCGAACCAACCAATATCTATCTGAC TTGTCTTCATCTTCTTCATCTTCCTCCTCCTCTTCCTGCTCCTCCTTCTTCATTGGAAGGGTTTTTAAT TACTGTTTCTGTTGCCTCCTTTGTTCTCAGTCTGTTGTTTTCTTTCTGGTTTAACTTTGTTGATTTGGA TGAATCAAAAAATCCATCCATTTCTTTTAGATTTTGCAATTTAATGGAGTACAGGTTTTAAAAAATATT TCATCATGGTATTCTGTATTTTTTCTAAAAAATATTTTCCTGTGCATTTCTAGTTCTGTTAATTTGGGT CCTCTTCTTTATTTTGGTTAGTTGAGCCAAGGGTCTATTGATCAAATTTATTTTCTCAAAGAACCAGCT CTTAGATCCACTGATTCTTTGTACTGTTCTTTTTGTTTCAATTTCATTAGTTTCTGCTTTGACTTTTTA GTATTTCTTGAGTTCCTATCCTGACTTCCTTTGATGATGAACAGTGATATGGAAGTATAAGTGGAATAA AT TAT TTTCTCTC TAAGT T GT T T T GAG C AT GGT GT T T CAT C AT AGCAGT AGT AACT C CAAAAC AAT T GA AATCATTATTATAGGAGGTAGCTGAGTTAGCTGGACTGCCAGCTCTCCTGCACCTGAACCACCAGGACA AATTCTCCAGCACTGCCCCAGGTAGCTCACCCAATGTTGGAGATGGCAAGGGGCATGAGCAGCTCCCTG CTGCGTAGTGGGCTCACCTTCTCCCTCCCCCACCACTATGGTCACCTCTACTTTGCTACCTAGGGCCTG CTCTCCCAGGTGCTGCTGTCCATGAGGGTCAGGATCAGCTCTCATGACCTCAGGGTCAGTTCTCTAGCC TGCCACAGGCAGATGAGGACAGCATCGCTCCTTTGCCCATGCCACCATGGTAGATGAGGGGTGGGACCA GCTCTCCCACACTCACAAGGTATGAATGTTTTAAGATGTTTGTAACATGGAAAAATTTTATTTCTCTTT CAACTAAGGCAGATAGTTTTGCTGAGTGCAGCAATCATGACATTTTAGAACCTGGAATGCCCAGCTCCA GGCTTTTCTTGCTTTCAGTTTGCACTAAGAAACCAGATGGGCTTCTCTTTATATGCCATATGTATGGTT TTTTTTCTCTTGCAGATTTCAGTATATTTTCTTTGTAGTGTCTATTTAAGTTTTAACTGTGTTATGCCA TGGGGTGTTTCTTTCCTGGTCTTGTTTAGTTGGTGGTTTGTGTCCTTATATCTGTATGGATGTGCCATT CTTCAGTCTGGGGAAGTTTTCTTCTATGTTTGTGTGAAAAATCTGATCTATGCTACTGACCTGGGCTTC TTCTCCCTGATTTTTCCTATAATTAGAAGATGAGATCCCACATTTCCAGCATATTCCTTTCCTTTGCTT TAAACATTTTCAAATATTTTGTTCTTCAAGTTCTGATTGTCTATCTTCTACTTGACTCATTTTATTTAG AAGTTAAGTTTTTTACTAATTGGTGTGTGTGTGTATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATA CATATTTGCCATAGTACCTATGTGGAGGTCAGATACAACTTGCAGGAGTCAGTTCTTTCCTTCCAAGAT GTGGGCCCTGGGCTTTTGTACTGAGGTTCTCAGGTGTGGCAGCAAGTGAGTGCTACCCACTGAGTAATC TTGTTGTCCCATAAATTGTGCTTTTTGGGTGTTTGGTCATTCAATGAAAGAACTGACACAATGCTATAC T T C AGAAT T C T T G AT AGAC AT AAGAAT G AAAT T T C T AAT AT T T T AAT T T T GT T GAAAT T GAT G T T AC AA GCAAAGAAAAAT AGACT AT AT T AT AGAAAAT AAT GACT T T T AGAACAC C C CT T AT ACAT C CAC ACAT GA CAAAAGATTTGGTGGAAATAGCTCAAAATATGGTATTTTTTGTTTCAATTTCCCTTTCATCTTTTTTCA AATACTCTGTAACTAGTGCACATTTTCTACACACCATTATGTTTTAGAAGATGCTTATTATTATCTGAG AAACAATTATTTTGTTTTGAAATTATGAATTGCAAGGCCCATATCTAAACATGATAAAAGCAATCTACA GCAAACCAGTAGC CAACAT CAAAGTAAAT GGT GAGAAGCT GAAACCAT CC CACTAAAAT CAGGGACTAG ACAAGGCTGTCCACTTTCTCCCTACCTATTCAATATAGTCTTTGAAGCCCTAGCCAGAGCAATTCGTCA ACAAAAGGAGAT CAAGGGGATACAAATT GGAAAGGAAGAAGT CAAAATAT CACTAT TT GCAGAT GAT AT GATAGTATATATAATTGACCATAAAATTCCACCAGAGAACTCCTAAACCTGATAAACAGCTTTAGTGAA GTAGCT GGATATAAAATTAACT CAAAAAAAGT CAAT GGCCT TT CT GTACACAAAGGATAAACAGGCT GA GAAAGAAATTAGGGAAACAACACCCTTCACAATAGTCACAAATAATATAAAATACCTTGGCGTGACTCT AACTAAGGAAGT GAAAGAT CT GTAT GAT AAGAAT GT CAAGT CT CT GAAGAAAGAAATTAAAGAAGAT CT CAGAAGATGGAAAAATCTTCCATGCTCATGGATTGGCAGGATCAATATAGTAAAAATGGCTATTTTGCC AAAAGCAATCTTCAGATTCAATGCAATCCCCATCAAAATTCCAACTCAATTCTTCAACGAATTAGAAAG GGCAATTTGCAAATTCATCTGGAATAACAAAAAACCTAGGAGAGCAAAAACTCTTCTCAAGGATAAAAG AACCTCTGGTGGAATCACCATGCCTGAACTAAAGCTGTACTACAGAGCAATTGTGATAAGAACTGCATG GTACT GATATAGC GACAGACAAGTAGAC CAAT GGAAT AGAAT T GAAGGCC CAGAAAT GAACCCACACAC CTATGGTCACTTGATCTTTGACAAGGGAGCTAAAACCATCCAGTGGAAAAAAGACAGCATTTTCAACAA ATGGTGCTGGCACAACTGGCAGATATCATGGAGAAGAATGAGAATTGATCCATTCTTATCTCCTTGTACAAAGGT CAAAT CT AAGT GGAT CAAGGAACT CCACATAAAAC CAGAGACACT GAAACTTATAGAGGAGAA AGTGGGGAAAAGTCTCGAAGATATGGGCACAAGGGGAAAATTCCTGAATAGAGCAGCAATGGCTTGTGC T GTAAGATT GAGAAT CGACAAAT GGGAC CT CATAAAATT GCAAAGCTT CT GTAAGT CAAAAGACACT GT CAATAAGACAAAAAGACCACCAACAGATTGGGAAAGGATCTTTACCAACCCTGAATCAGATAGGGGACT AAT AT C C AAT AT AT AT AAAGAAC T C AAG AAG GT G G AC T C C AGAAAAT C AAAT AAC C C CAT T AAAAAAT G GGGCT CAGAGCTAAACAAAGAATT CT CACCT GAGGAATACC GAAT GACT GAGAAGCACCT GAAAAAAT G TTCAGCATCCT T AAT CAT C AGAGAAAT G CAAAT C AAAAC AAC C C T GAGAT TC CACCT CACACCAGTTAG AGT GGCT AAGATAAAAAATT CAGGT GACAGCAGAT GCT GGC GAGGAT GT GGAGAAAGAGGAACACT CCT CCATTGTTGGTGGGATTGCAAGCTTGTACAACTATTCTGGAAATCAGTCTGGCGGTTCCTCAGAAAATT GGACATAGTACTACCGGAGGATCCCGCAGTACCTCTCCTAGGCATATATCCAGAAGATGTTCTAACCGG TAAGAAGGACACATGCTCCACTATGTTCATAGCTGCCTTATTTTTAATAGCTGGAAGCTGGAAAGAACC CAGAT GT CC CT CAACAGAGGAAT GGATACAGAAAAT GT GGT ACATTTACACAAT GGAGTACTACT CAGC TATTAAAAAGAATGAATTTATGAAATTCCTAGGCAATTGGATGGACCTGGAGGGTATCATCCTGTGTGT GGT AAC C CAAT CACAAAAGAAC T CACAT GAT AT GT ACT CAC T GAT AAGT G GAT AT T AGC C CAGAAACT T AGAATAC CCAAGATACAAGATACAATTT GCAAAACACAT GAAACT CAAGAAGAAAGAAGAT CAAAAAGG TGTGGACACTTTGCCCCTTTTTAGAATTGGGAACAAAACACCCATGGAAGGAGTTACAGACACAAAGTT TGGAGCTGAGACGAAAGGATGGACCATTTAGAGACTGCCACACCCGGGGATCCATCCCATAATCAGCCT CTAAACGCTGACACCATTGCATATGCCAGCAAGATTTTGCTGAAAGGACCCTGATATAGCTGTCTCTTG TTTGGCTATGCCAGTGCCTGGTAAACAAAAAAGTGGATGCTCACTGTCAGCTATTGGATGGATCACAGG GCCCCCAATGGAGGAGCTAGAGAAAGTACCCAAGGAGCTAAAGGGATCTGCAACCCTATAGGTGGAACA ACAATATGAACTAACCAGGACCCCCCCAGAGCTCGTGTCTCTAGCTGCATATGTATCAGAAGATAGCCT AGTCAGCCACCATTGGAAAGAGTGTCCCCTTGTTCTTGCAAACTTTATATGCCTCAGTACAGGGGAACA CCAGGGCCAAGAAGTTGGAGTGGGTGGCTAGGGGAGTGTGTGGGGGAGGGTATGGGGGACTTTTGGGAT AGCATTTGAAATGTAAATGAAGAAAATACCTAATAAAAAAAGAAATTATGAATTGTAAGCACTTAAAAA TATTTAAAGAGAAAGTTGTGTTTATGGAACATTCATTTACTTTGCTAGTACAATTGCTAGTTGCTCTGA GAGATATGTGAAGGAAAAACAACTCTTTTTACTTTCTAGATATGTTCCTATTTCACCTATTGGCAGAAC TAATAGGAAAAACTT T GCACACAATATATTACAAATAT GGT T GGAGGTAAGAGAT GAGTT GCACTAT GA GCCATGAGGGAAGAGAGTCATTAAGGCTTGGCATAGTCTTGGAACAATGTTAACCAAATGGAGATTTTC ACACTCAGAGAGTTAGAATCTTCTAGAATTATGCAAGCCAGACAAAGAGGAAGAATCATTAGGGAAGTC TAAGAATATTGCCTTTCCTTATGCATGCTGCTGGGCACCAGGGCAGGACTGTGTGTGAGATGAGGCACT ATGGGAGCAGAGGGTCTTGACCTGCGTGCTGTAGGGTCTGGGTTAGTGGGCACAGCTTTATTCATTGAG CAGTTCTTTCTGTGTTAGATTATAGTTCAGCTTGTTCCTTCCTTCTTGTTTGGAGGGCTTCTCCTCTGC TAGCATGGGTTTTTTTTCTTTCTTTCTTGTAATCAGCTGATTGGTGTTTTTAAATTGAATGGCCTCTAT ATTCAAAACATTCAAAGGGAGAGCAAAAGGCTGTTATTGAATAAGGTGAGGCAGAGAGGCAAGGAAGTA CAGGTGGATAAAAGTAAATATTATATCCTGTGGTAAAATGGTGATTATTTTACATTTCCATTTAGGGGT GAGCTGTTAGCCTGAGTCACACTGAAACTTTAAGGTTTAATGAAGTAGAAACAGACTTGTCTGTCCACC CCCCCCCTTGTCTGTTTTAAGGTCAACATTTTTGGTTCCCTTTTTGGCTCTTAGTAACGACTTTTGGTT TCATGAGTACATGGTTGTTTTAGTCAGGGTTTCTATTTCTGCACAAACATCATGACCAAGAGGCAAGTT GGGGAGGAAAGGGTTTATTCAGCTTACACATCCATACTGCTGTTCATCTCCAAGGAAGTCAAGACTGGA ACTCAAGCAAATCAGAAAGCAGGAGCTGATGCAGAGGCTATGGAGGGATGTTCTTTACTGGCTTGCTTC CCTGGCTTGCTCAGCTTGCTTTCTTATAAAACCCAGGACTCCTAGCCCAGGGATGGCACCACCCACAAG GGGCCCTTCCCCCTTGATCTCTAGTTGAGAAAATGCCTTACAGATGGATCTCATGGAGGCATTCCTCAA CTGAAGCTCCTTTCTCTGTGATAACTCTAGCTTGTGTCAAGTTGACACAAAACCAGCCAGTACAATGAT AAATTATATTTTGAAGGTGGTAGTTGAAACTGGATTAAATGTGACAAGAGGAACAGATAGGAATGCCTA AATAAACAATAAAAGTAACTCAAAACTCTCTTTAAAGATGTGTTAAATGCCTGGGCATGGGCACCCCTT CATTCCAGCTCGGGAGGGAGAGGCATGTGGACCTCTGTAAGTTTGAGGCCAGCCTGTTCTATGAGATGA GT T CT AG GACAT T CAGT GAT GT T ACAGAGAGAAT CCCTGTCT T AAAAAAACAGAAC AAACAAAT GAACA AACAAAC AAAACAGC AACAACAAAAAC C AAC CAAC CAAC CAAC CAAACAAC CAAC C AAC CAAC CAAAAC C CAC CAC CAC CAC CAC C CACAAAAAGT AT T AAAT T T GT TAG T CT GAT CT GAACAGC CAAAT T T AAAT GT GTTGTCGACATCTTTAGATGATCCAGAAACCTGGAGGAAGAGTCTCCATGATAATGCCTGCCATGTCTA ATTCTCATAAGATCAGCTGGACCCTGCGCATCATCATTCTACTTCACCATGCACAGAGTGAGAAGCCCA AGCTTTTCTCAGAGAACTGACTTGTGCCTCTGGAGATGCTGCCACAGATGGTATGTATGGGCCTCTGGA CTGTCTTATGTTCTGTTACCTTCAGATATGATTTTATTCTGTATAAGTTGTCTTCTTGGGAAATGGAGG TCATACCTAGCTACGTTAGAATCAGCAGAGGGCAACTGTTAGGTTCCGTCATCCACTGTAGAAGTGGGTAGCAGTAGCAGGGTGGAGATTCGAGAGCTAAGGTAGATGCTGTTGTCAGAACCTGTAGCGCATGCCCCT TAACTTTAGACTTCACAAGCTTTGTGGACATCTTAGGCAGGACACAGAATGAGCAATGTATTTAATGAA AGGAACTCATGCCTGTTACGTAGGCTTGGAGAAGCACCCTGTACTAATAGGAAGAAGCATCATCTACAG GAACAGTTTTCATTCTTTGTACTATGTTCTCAAGTTTAGTAGATTTTCTTTTGGTTACATTCTTCTTTT T ACACT T T G CAT GAC AAAT GAC AGAGAT AT TAT CT CACAGAT GT GAAT GT AC CAAC AT AT AGAGACAAA AAGGCCAAAGCCAAAGGGAAAAATTTTAATACCGTAACTCCACCACAAAACAATAAGAATTTACATTAA T T T AAAAAAAAAC CT ACAAGAC AGCAAAAAAAACAAAACAAAAGAAAACAAAAAAC C GAAGT C C C CT AC AGAGGAAACTTACTCAGTTGGCAGTTGTTGGATCATTACTGCTTGGCGCCAGACCCAGTAACTTAGAAC ACCTTTTTAATCAGGGTCTCATTCCTAATGGTCCCCAACGAGGCAAATGATGTTATATCAAACTGACTA CCTCAAGATAGGGAGCTGCCATCTCAAACTTTGTTCTGATGCATCTCAATGTGAATGGCTCACAGGGCA AGCTCTCTTAACTGTTATGAAAGAACCACATTATTCCTTTTCATCTCTGGGACTCTGAGCATGAGACCT TGCCCACTATGTTTTTGTCCACAGTGTCTGTTCTTCACTAAAACTTTTATATCAGTTCTGGTTTGATAA ATACTTCTATCTCTGTGCCTTGGGCTCATCACTCTTGGCATGATACTATTTGCCAAAATGACTGTGCTA C C CAT T CAT TAT G GT GAAACAC AAAGT GAACAT T T GAT AGT ACAAGT AT T CACT GT GAAACT T T T GGGT AAAGCTAGTGTTATTGTTGTTTTAAATATGACTGAAGTATCAAACTGTAGGGTGAATGGGAACTTAGGC CATCAGCCGGCAGCACAGTCTGTAGGGTGAGAGGGAACTTAGGCCATCAGCGGGCAGCACAGTCTGCAG TGGGCCAGGGCAGGTGCTTCTGAACACAAGGCTAGGAAACATGGTGCTTTCCTCTGCATCCCTGCACAC CTCTCACAGGAAGAGTAAGTGGCGGTGTGAGCTTACATGCATTAAAGAGCCAGTGACACTACAGCCAAC CAGTAAACGTTTACTAGGAATCTCCTATTGGTAAGACATTATACTGGAGTCAAGGGCAAAGTTAATTTT CTCAGGAAAGTTTCACCAAATTTCTGGGGTCATATAAAATCTATTAAAGGAACATTTTCTCAAGACAGT CTTTAGGAATAGAAAGTAACATTGTAGGTAATTTAGAAAAAAAATGTACACCAAGACTCAAAACAGGAA AT AT T C AGAT AAC AG G C C AAAAT G C C T T T AAT AAC AGT AAAAAT AAT AT C AAT AAAAC T C AAAT AT T T T CTAAGCAAGACAATATAGAAATATCTACTTTACAAAATAATTTACACTATTTAATCATGATACCTGGAA GTT GT GT CT GGGAAAAAT CTACAAGGAT AT GAAGCATAAACATAATAGT CATAATATATAAT GGTACAA T AAT T T ATACT GAAC T AGT AGAGAAAAC T AAACAT T GAGT G CT T TAT CT G CAT AAG GT AGT AT T T CAAG AAATGTCCTCTGCAGATCTTGGAGATTGAAAATTGATGACACAGTACAGTGAGAGTCTTTTTTCCTAGA TGCTCAGTCCCAGAGCGACAATTTCTTCTCTTCTTCATTTTCCACTTTCCTAAGCTCTATGATCTATCT GGTGTCTAGGCTGTCTTCCATTCTCCTAGAATGTGCTTTTACAACCAGGGTGTCTATGTGGAGAGGTCA T GAGAAAAT TAACAACAAAAGCAGCTAT TT CAGAC GT GTTAT CTACAGT GAT GGTT T GGCT GAGAAAAA GAGAGT AGAAT T T GC AGAGT GGAT T CAGAGAGT GAAGGT GAT GT AT AGT C T T AAC G CAC GAAAACT GT A T AT AT AAAG T GAG GG AAAAAGAAAT T T AAC AAAAAAT AGAG AG GAAAG GT T AAT T T T G GT AAAAGAT C A AAGTTCCACATTTGGAGACAGAACAGAAAATTGAAGGGAAAGGAAATCATGAAGGGGTCATCAGACATA AGGGACT GC CAGT GGT GCCAGT CAGTAT CT CAGCAT GT GAGAAACGACGGACAGAGAGAGAAT GT GAGG GACACCGAGCTGCATGGAGAAGGAAGCAGAGAGAAGAGAGCAAGGAGACAGTGGGAGGAAACATGCCAA AGAGACTCTTAACTAGAAATAGAACACACAGAAACAACATACAGGAAAGAGTGCATAAGGACCATACTG GTGTGCAGATCTCTGTATGAAAATGGAAAACTCAACAGCCTGCTAGACGTGCAAAACCAGAGAGCATTG TAAGTAATTTGAGAGAAACA ( SEQ ID NO : 19 )Example 2Materials and MethodsExperimental Animals and Ethics Approval

[0123] J-DTR mice were originally generated fee-for-service by InGenious Targeting Laboratory. Upon receipt, animals were quarantined and verified pathogen and parasite free before being released for use. Animals were subsequently bred and maintained at the USask Lab Animals Services Unit. WT and Jchain+IDTRheterozygote female and male mice were utilized for all experiments. Animals ranged in age from 3-7 months when used for experiments. Animal care and use were conducted according to the guidelines of the USaskUniversity Animal Care Committee Animal Research Ethics Board (Animal Use Protocol (AUP) 20220020).Creation of J-DTR mice

[0124] This mouse model was created using a genetically engineered mouse embryonic stem cell line, in which a custom targeting vector was designed so that the internal ribosomal entry site (IRES)-D R cassette was inserted after the TAG stop codon of the Jchain gene. The knock-in cassette was followed by an FRT-flanked neomycin (Neo) selection cassette. The long homology arm (l_A) of the vector is ~6 kb in length and the short homology arm (SA) is ~2.1 kb in length. The region used to construct the targeting vector was subcloned from a positively identified C57BL / 6 BAC clone using homologous recombination-based techniques. The targeting vector was confirmed by restriction analysis and sequencing after each modification step.

[0125] The targeting vectorwas then linearized and electroporated into a FLP C57BL6 (BF1) embryonic stem cell (ESC) line. After selection with G418, antibiotic-resistant colonies were picked, expanded and screened via PCR analysis and sequenced for homologous recombinant ESC clones. The Neo resistance cassette was removed via FLP recombinase in the ESCs during expansion. Positively targeted ESC clones were then microinjected into BALB / c blastocysts and transferred into pseudo-pregnant females. Resulting chimeras with a high percentage black coat color were mated to C57BL / 6N WT mice, after which the offspring were tail-tipped and genotyped for germline transmission of the targeted allele sequence. Germline mice were identified as heterozygous for the co-expression of the DTR cassette in the mouse Jchain gene locus. Upon receipt, mice were bred to eliminate the gene encoding the flippase (i.e. , FLP) recombinase.Genomic DNA Isolation and Genotyping

[0126] Ear biopsies were incubated at 100 °C in 400 mL of 50 mM sodium hydroxide (NaOH) until tissue was fully dissolved. NaOH was neutralized by the addition of 1 M Tris- hydrochloric acid (HCI), pH 8.0 (50 mL). Samples were vortexed and then centrifuged at 25 °C and 12,000g for 2 minutes. Supernatant (200 mL) was transferred to a new 1.5-mL tube. After the addition of 3 M sodium acetate (NaOAc), pH 5.2 (20 mL) and 95% ethanol (EtOH) (660 mL), samples were vortexed and DNA was precipitated overnight (O / N) at -20 °C. Thenext day, samples were centrifuged at 4 °C and 12,000g for 5 minutes. Supernatant was aspirated and DNA pellets were resuspended in 100 mL of 0.1x Tris-EDTA buffer.

[0127] For genotyping PCR, each reaction consisted of 10 mL Platinum II Host-Start PCR 2x Master Mix Thermo Fisher Scientific, Cat# 14000012), 1 mL forward primer, 1 mL reverse primer, 2 mL DNA and 6 mL H2O. All primers were resuspended at a concentration of 1 mg / mL in 0.1x TE. Reactions were amplified using a Veriti 96-well thermal cycler (Thermo Fisher Scientific). Reactions products were electrophoresed in 2% agarose gels containing ethidium bromide and products were visualized under ultraviolet light using a BioRad ChemiDoc Imaging System.In Vivo DT T reatment

[0128] 1 mg of lyophilized DT from Corynebacterium diphtheriae (Millipore Sigma,Cat# D0564) was resuspended in 0.5 mL sterile H2O yielding a 2 mg / mL DT concentration in a 10 mM Tris-1 mM EDTA, pH 7.5 solution. For injection, DT was subsequently diluted to 2 mg / mL in 1x phosphate buffered saline (PBS) (Gibco, Cat# 21600-069). Mice received 100 mL i.p. injections of either PBS or DT (200 ng total).Isolation of Bone Marrow, Spleen and Thymus Tissue

[0129] All tissues were processed and collected in calcium and magnesium-free 1x PBS. SPL and THY were dissected and crushed between the frosted ends of two slides. BM was isolated from both femurs and tibias by cutting off the end of bones and flushing the marrow from the shafts and ends using a 23-gauge needle. Cell suspensions were centrifuged for 5 minutes at 4 °C and 600g. Red blood cells were lysed by resuspending cells in 3 mL of 1x red blood cell lysis buffer on ice for ~3 minutes. Lysis was stopped with the addition of 7 mL of 1x PBS. Cell suspensions were strained through 40 m filters and counted on a Countess 3 (Thermo Fisher Scientific) using Trypan Blue to exclude dead cells. Cell suspensions were centrifuged as before (5 minutes at 4 °C and 600g) and resuspended at 2x107cells / mL in 1x PBS + 0.1% bovine serum albumin (BSA, Fisher BioReagents, Cat# BP9706-100) before use.Isolation of Fecal Pellet Supernatants

[0130] Fecal pellets were collected from dissected mouse intestines and colon. Tools were used to open the organs and collect fecal pellets into 1.5-mL microtubes. The collectedpellets were placed on ice until generation of fecal pellet suspensions. To generate suspensions, pellets were weighed and 1x PBS was added at 1 mL per 1000 mg of feces. Tubes were vortexed at max speed in a 4 °C cold room for 10 minutes. The fecal suspensions were centrifuged at 12,000g for 10 min at 4 °C. Then the supernatant was transferred into a new microtube and stored at -80 °C until use.Isolation of Plasma

[0131] Blood was initially collected into 1.5-mL microtubes via cardiac puncture then placed on ice until completion of tissue harvesting. Subsequently, blood was set at room temperature (RT) for 30 min and then centrifuged at 1000g for 10 min in 4°C. Plasma was transferred into a new microtube and stored at -80 °C until use.B cell and ASC enrichment

[0132] EasySep Mouse Pan-B cell Isolation and EasySep Release Mouse CD138 Positive Selection kits from STEMCELL Technologies were used to enrich B cells and ASCs from ~5x107SPL cells following manufacture guidelines. Isolated cells were collected in a final volume of 1.5 mL of 1x PBS + 2% fetal bovine serum + 1mM EDTA and counted using a Countess 3 with Trypan Blue to exclude dead cells and calculate final yield.Quantitative polymerase chain reaction (qPCR)

[0133] RNA was extracted from isolated B cells and ASCs using the PureLink RNA Mini Kit (Thermo Fisher Scientific, Cat# 12183025). RNA was quantified using a NanoDrop Onec(Thermo Fisher Scientific) and verified to have a A260 / 280 ratio of ~2.0. The Maxima H-Minus First Strand cDNA Synthesis Kit with dsDNAse (Thermo Fisher Scientific, Cat# K1682) was used to generate cDNA. Each cDNA synthesis reaction included >10 ng RNA mixed with 1 JJL random hexamers primers, 1 JJL 10mM dNTP mix, 4 JJL RT buffer, 1 JJL Maxima H-minus enzyme mix and the appropriate volume of water required to obtain a 20 |iL reaction. The reaction mixtures were incubated in a Veriti 96-well thermal cycler using the manufacturer recommended amplification program. qPCR was performed using a StepOnePlus Real-Time PCR System (Applied Biosystems). Each 20 mL reaction contained 2x TAQMAN Fast Advanced Master Mix (10 JJL), 20x TaqMan primer (1 JJL), cDNA (2 JJL) and water (7 JJL). Triplicate reactions were run 96-well plates using standard TAQMAN amplification conditions. All primers are listed in the Key Resources Table. Expression fortarget genes was calculated as 2(ActbcT> Tar9etcT)and represents the average derived from triplicate technical replicates.Immunostaining

[0134] All staining procedures were performed in 1x PBS + 0.1 % BSA. Samples were labeled with a CD16 / 32 Ab to eliminate non-specific binding of Abs to cells via Fc receptors. All Abs utilized are listed in the Key Resources Table. Cells were incubated on ice for 30 minutes in the dark with the appropriate Abs. Unbound Abs were washed from cells with 1x PBS + 0.1 % BSA followed by centrifugation for 5 minutes at 4 °C and 600g. Supernatants were decanted, and cell pellets were resuspended in an appropriate volume of 1x PBS + 0.4% BSA + 2 mM EDTA for flow cytometric analysis. Before analysis, cells were strained through a 40 jim filter mesh and kept on ice in the dark. eBioscience Fixable Viability (Live- Dead) Dye eFluor 780 (Thermo Fisher Scientific, Cat# 65-0865-14) was added to samples to assess dead cell content. The stock solution was diluted 1 :250 in 1x PBS and 10 mL was added to ~5 x 106cells per stain. Live-Dead stain was added concurrent with surface staining Abs.Flow Cytometry

[0135] Flow cytometry was performed on a CytoFLEX (Beckman Coulter) located in the Cancer Cluster at USask. Total cells were gated using side scatter area (SSC-A) versus forward scatter (FSC-A) area. Singlets were identified using sequential gating of FSC-height (H) versus FSC-A and SSC-H versus SSC-A. All data were analyzed using FlowJo (v10) software.Enzyme-Linked Immunosorbent Spot (ELISpot)

[0136] ELISpot plates (Millipore Sigma, Cat# MSIPS4W10) were briefly incubated at RT for l minute with 15 |iL of 35% EtOH. EtOH was removed and wells were washed 3 times with 150 |iL of 1x PBS. Subsequently, wells were coated O / N at 4 °C with 100 JJL of capture Ab. The capture Ab (Millipore Sigma, Cat# SAB3701043-2MG) recognized mouse total IgG+IgM+lgA isotypes and was pre-diluted in 1x PBS to a final concentration of 5 jig / mL before use. The next day, coating Abs were removed, and wells were washed 3 times with 150 |iL RPMI 1640. Plates were subsequently blocked with 150 JJL RPMI for a minimum of 2 hours at 37 °C in a 5% CO2 / 20% O2 tissue culture incubator. Blocking solution was removedand total cells from BM, SPL and THY were deposited into wells with a target number of 105cells per well in a 100 mL volume (3 wells per sample). Cells had previously been resuspended at 1x106cells / mL in RPMI supplemented with a proliferation-inducing ligand (APRIL) (10 ng / mL), interleukin (IL)-6 (10 ng / mL), heat-inactivated fetal calf serum (10%), Penicillin-Streptomycin (100 U / rnL), L-glutamine (2 mM), Gentamicin (50 jig / mL), sodium pyruvate (1 mM), non-essential amino acids (1x), non-essential vitamins (1x) and 2- mercaptoethanol (10-5M). Cells were then incubated O / N (>12 hours) at 37 °C in a 5% CC>2 / 20% O2 tissue culture incubator. The next day, culture supernatants and cells were removed. Wells were washed 3 times with 150 JJL of 1x PBS then an additional 3 times with 150 |iL of 1x PBS + 0.1% Tween-20 + 1% BSA. Secondary Abs conjugated to horseradish peroxidase (HRP) were added at a volume of 100 JJL per well and plates were incubated for 2 hours at RT. Anti-IgM-HRP (Thermo Fisher Scientific, Cat# 62-6820) and anti-lgA-HRP (Thermo Fisher Scientific, Cat# 62-6720) were both diluted at 1 :1000, and anti-IgG-HRP (SouthernBiotech, Cat# 1015-05) was diluted at 1 :50,000. All secondary Abs were diluted in 1x PBS + 0.1 % Tween-20 + 1% BSA before use. Following incubation, Abs were removed and plates were washed 3 times with 150 JJL of 1x PBS + 0.1 % Tween-20 + 1% BSA followed by an additional 3 washes with 150 JJL of 1x PBS. To reveal “spots”, 100 JJL of Developing Solution from the AEC Substrate Set (BD Biosciences, Cat# 551951) was added to each well. Plates were shaken at 200 rpm for 30 minutes at RT. Developing Solution was removed and plates were washed 5 times with 150 JJL of H2O. Well backings were removed, and plates dried at RT O / N after which “spots” were visualized with a Mabtech ASTOR ELISPOT reader. Additional wells lacking capture Abs were developed to gauge background. For spot quantification, counting was restricted to a 1250-pixel area of interest to avoid edge artifacts. For each genotype and treatment combination, 3 background wells were counted, averaged and then subtracted to obtain the reported spot numbers.Enzyme-Linked Immunosorbent Assay (ELISA) for blood plasma and fecal pellet supernatants

[0137] High binding ELISA plates (Greiner, Cat# 655081) were coated using 100 JJL anti-mouse IgG / lgA / IgM (H+L) (Sigma Aldrich, Cat# SAB3701043-2MG) at 5 jig / mL in 1x PBS per well and incubated at 4 °C O / N covered with plastic wrap. Coating Abs were removed and plates were washed 3 times with 150 JJL 1x PBS + 0.1 % Tween 20 (WashSolution) per well. Subsequently, 150 jiL of 1x PBS + 1 % BSA + 0.1 % Tween (Block Solution) was added per well and plates were blocked at RT for 2 hours. Block Solution was removed and wells washed 3 times with 150 mL of Wash Solution. Purified Ab standards (Standard Curves) and plasma or fecal supernatant samples were diluted at various concentrations in 1x PBS and 100 mL per dilution was added per well. Plates were incubated at RT for 2 hours; samples / standards were removed and wells washed 3 times with 150 mL of Wash Solution. Isotype-specific HRP-conjugated secondary Abs were diluted in Block Solution and 100 mL were used per well. Secondary Abs for IgM and IgA were diluted to a final concentration of 1 :5000 while those for IgG were diluted to 1 :50,000. Following a 2-hour RT incubation, wells were washed 3 times with 150 JJL of Wash Solution, then incubated for 4 minutes with 100 |iL 1x TMB Substrate (Thermo Fisher Scientific, Cat# 00-4201-56) per well. Enzymatic reactions were stopped with the addition of 100 JJL per well of 0.16M H2SO4 (Fisher Chemical, Cat# SA431-500). Optical densities (ODs) were read at 450 nm wavelength using a BioLegend Mini ELISA Plate Reader (BioLegend, Cat# 423555). Samples and standards were analyzed following subtraction of blank wells (1x PBS) and assayed in triplicate. Plasma and fecal supernatant Ab concentrations were calculated using linear portions of standard curves and the equation of a straight line (y = mx + b) where y = average OD per sample, m = slope, x = antibody concentration and b = y-axis intercept. Only experiments with a linear standard curve R2> 0.98 were considered valid.Western Blotting

[0138] ASC-enriched samples were generated as indicated in Section 2.8. After counting, cells were centrifuged then lysed using the Thermo Fisher Scientific Cell Extraction Buffer (Cat# FNN0011) following the provided protocol. The only protocol exception was that cells were concentrated 10x in terms of lysis buffer volume. Lysates were stored at -80 °C until use. For western blot analysis, sample lysates were combined with Bolt Sample Reducing Agent (10X) (Thermo Fisher Scientific, Cat# B00040) and Bolt LDS Sample Buffer (4X) (Thermo Fisher Scientific, Cat# B0008) then loaded into a Bolt Bis-Tris Mini Protein Gel, 4-12% following the manufacturer’s protocol (Thermo Fisher Scientific, Cat# NW04122BOX). The PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific, Cat# 26619) was used as a size standard. Following the Thermo Fisher Scientific Mini Blot Module electrophoresis and blotting protocol, the gel was electrophoresed and proteins transferred to a PVDF membrane using Invitrogen PVDF / Filter Paper Sandwiches 0.45 mm (Cat#LC2005). After transfer, the PVDF membrane was washed with nuclease-free H2O (Thermo Fisher Scientific, Cat# AM9906) and transferred proteins were revealed following staining with 0.1% Ponceau S / 5% Acetic Acid for 15 minutes. A cell phone camera image was taken of the Ponceau stained membrane protein. The membrane was washed with Milli-Q H2O 3x at 5 minutes per wash then blocked with 1x PBS + 1% Tween + 5% nonfat dry milk (Membrane Blocking Solution) for 1 hour at RT. The membrane was cut above the 35 kDa marker as Jchain possesses a predicted molecular weight of 18 kDa and the bottom third of the membrane was incubated O / N at 4 °C with a rabbit anti-human / mouse / rat IgJ (Jchain) (Thermo Fisher Scientific, Cat# 13688-1-AP) primary Ab diluted 1 :250 in Membrane Blocking Solution. The following day, the membrane was washed 3x with 1x PBS + 1 % Tween before application of a goat anti-rabbit IgG-HRP secondary Ab (Southern Biotech, Cat# 4030-05) diluted 1 :10,000 in Membrane Blocking Solution. Following a 2-hour 400 revolutions per minute shaker incubation at RT, the membrane was washed 3x with 1x PBS + 1 % Tween. The membrane was developed using a freshly mixed Novex ECL chemiluminescent substrate reagent kit (Thermo Fisher Scientific, Cat#WP20005) for 1 minute before being imaged with a Bio-Rad ChemiDoc using the Chemiluminescent program.Quantification and Statistical Analysis

[0139] The numbers of mice used (n =) per experiment are listed in the Figure Legends. Statistical analyses were performed using GraphPad Prism (v8.4.2) software. Quantification of cell numbers and various flow cytometry data are graphically represented as mean ± SEM. Statistical analyses are described within each Figure Legend and statistically significant p-values are shown within each Figure.ResultsThe generation of J-DTR mice and validation of DTR gene expression in ASCs

[0140] DT is derived from Corynebacterium diphtheria and acts as a potent protein synthesis inhibitor leading to cellular apoptosis

[0019] , Previous work demonstrated that DT enters the cell via receptor-mediated endocytosis following binding to the membrane bound pro-form of heparin-binding EGF-like growth factor (HB-EGF protein, encoded by the HBEGF gene) [20,21], While species such as humans, simians and mice all express the HB-EGF protein, the mouse version of the protein possesses distinct amino acid differences making this species relatively insensitive to DT

[0022] , As such, mouse models have been developedin which human or simian HBEGF (referred to throughout as the DTR) expression is driven by cell type-specific genetic elements thus allowing for targeted ablation of that particular cell type [23-25],

[0141] In considering an appropriate driver of DTR in ASCs, the expression of various ASC-associated genes in the Immunological Genome Project database (ImmGen, https: / / www.immgen.org / ) were examined (FIGs. 16A-16C). The ASC-associated transcription factor Prdml (BLIMP-1) and cell surface marker Sdc1 (CD138) were readily expressed by ASC subsets; however, this was not exclusive as both genes were found to be expressed in other cell types albeit as lower levels (FIG. 16A). Recent work utilized the Jchain gene to drive a CreERT2 cassette in ASCs with a great deal of success

[0011] . Jchain expression was increased in ASC populations compared to both Prdml and Sdc1 upon examination of the ImmGen data (FIG. 18B). Furthermore, Jchain expression appeared highly selective for ASCs when compared to all ImmGen cell types (FIG. 16B) as well as those specifically in the B cell lineage (FIG. 16C). Therefore, a C57BL / 6 mouse strain was generated with the DTR cDNA from Chlorocebus sabaeus (a.k.a. African green monkey) knocked into the endogenous Jchain locus (FIG. 1 A). In this instance, DTR was inserted into the Jchain 3’ untranslated region (UTR) downstream of an internal ribosomal entry sight (IRES) (FIG. 1A). Upon extraction of genomic DNA, both wildtype (WT) and DTR-inserted Jchain alleles were readily identifiable by polymerase chain reaction (PCR) (FIG. 9).

[0142] Next, to validate that DTR was expressed transcriptionally in ASCs, splenocytes from female and male Jchain+I+(WT) and Jchain+IDTR(J-DTR) mice (3-7 months old) were harvested. Using Pan-B and CD138 (ASC) selection kits from STEMCELL Technologies, enrichment of splenic B cells (CD19+CD138' / LO) and ASCs (CD138HICD267(TACI)+) as confirmed by flow cytometry (FIGs. 2A-2B and FIG.10A) was conducted. cDNA was subsequently generated from these populations and quantitative polymerase chain reaction (qPCR) analysis showed that ASC enriched samples possessed significantly higher Prdml (FIG. 10B) and Jchain (FIG. 10C) gene expression. However, only ASCs enriched from the SPLs of J-DTR mice showed high levels of DTR gene expression (FIG. 10 D). Due to variability in the effectiveness of ASC enrichment, we also examined DTRex. pression when normalized to that of Prdml (FIG. 10E). ASCs from J-DTR SPLs again displayed increased levels of DTR transcripts when compared to WT ASCs (FIG. 10E). In contrast, Jchain transcripts were equivalent between ASCs from the SPLs of WT and J-DTRmice (FIG. 10F). Western blot analysis of ASC-enriched SPL protein lysates further demonstrated that insertion of DTR cDNA did not ablate Jchain expression (FIGs. 17A-17B)

[0143] Finally, it was confirmed that the DTR protein could be found on the surface of ASCs from J-DTR mice. ASCs from the SPL (FIG. 11 A), BM and THY were identified as CD138HIlgD / LOCD90.2 / LOCD267(TACI)+CD44+similar to previously published data

[0026] . As shown in FIG. 11B, DTR expression was readily observable on the surface of ASCs from the J-DTR SPL compared to SPL ASCs or B cells (CD19+CD138 / LO) from both genotypes. Quantification of the DTR geometric mean fluorescence intensity (gMFI) demonstrated that J-DTR ASCs from the SPL (FIG. 11C), BM (FIG. 11 D) and THY (FIG. 11E) had significantly higher DTR expression compared to their WT counterparts. ASCs include 2 major populations: 1) proliferative, relatively immature and short-lived PBs and 2) post-mitotic, mature PCs with long-lived potential. In mice, CD45R(B220) expression can be used to delineate between these 2 populations (FIG. 11A) [9,10,26,27], In all 3 organs examined, DTR levels were increased in PBs and PCs from J-DTR mice compared to cells from WT animals (FIGs. 11F-11H). Notably, increased DTR expression was observed in PCs from the J-DTR SPL and THY relative to the PB compartment (FIGs. 3F, 3H). Investigation of whether DTR levels were uniform amongst ASC populations expressing different Ab isotypes was conducted. Using flow cytometry, membrane IgM (mlgM)+, membrane IgA (mlgA)+and double negative (DN) ASCs in the SPL, BM and THY were identified (FIG. 18A). Compared to WT ASCs, all 3 ASC Ab subsets from J-DTR mice possessed increased amounts of DTR (FIG. 18B). J-DTR mlgA* ASCs had significantly higher DTR expression compared to rnlgM* and DN ASCs regardless of organ analyzed (FIGs. 18C-18E). In both the SPL and THY, mlgM* ASCs from J-DTR animals displayed intermediate levels of DTR compared to mlgA* and DN ASCs (FIGs. 18C and 18E).

[0144] To determine if DTR was preferentially expressed in ASCs compared to other B cells types, DTR surface expression on total B cells (CD19+CD90.2' CD138 / LO) was examined as well as germinal center (or germinal center-like) B cells (GCB, CD19+CD90.2' CD138- / LOCD95(Fas)+GL7+)from both the SPL (FIGs. 19A-19B) and THY (FIGs. 19C-19D). As a comparison within the same flow cytometry samples, CD138HICD90.2- compartment was analyzed which would be enriched for ASCs. As expected, total B cells demonstrated minimal DTR expression while ASC-containing CD138HICD90.2' cells possessed high levels of the protein (FIGs. 19E-19F) in J-DTR animals. Within the SPL, J-DTR GCBs possessedan intermediate phenotype while the GCB-like population in the THY seemingly lacked DTR expression (FIGs. 19E-19F). Without being bound by theory, these data suggest that while all ASCs from J-DTR mice preferentially express the DTR, some organ-specific differences may exist based upon maturation status.J-DTR mice demonstrate normal generation of ASCs in the SPL, BM and THY

[0145] The above studies validated DTR expression by ASCs from the SPL, BM and THY. However, it was unclear if DTR expression via insertion into the Jchain locus altered homeostatic generation of ASCs or secretion of various Ab isotypes. Flow cytometric quantification of total ASCs in the SPL, BM and THY showed no significant differences between WT and J-DTR mice (FIG. 12A-12C. In terms of Ab secretion, enzyme-linked immunosorbent spot (ELISpot) assays demonstrated the presence of IgM, IgG and IgA ASCs in SPL, BM and THY (FIG. 12D). Quantification of spot numbers for IgM (FIG. 12E), IgG (FIG. 12F) and IgA (FIG. 12G) showed no significant alterations between WT and J-DTR mice in the SPL, BM or THY.

[0146] Enzyme-linked immunosorbent assay (ELISA) assessment of plasma Ab isotypes demonstrated concordant results with plasma IgM (FIG. 12H) and IgG (FIG. 121) being similar between WT and J-DTR mice. In contrast, IgA was significantly elevated in J- DTR plasma (FIG. 12J). While IgA is relatively rare in the plasma, it is present in high amounts in the feces

[0029] due to transport across the intestinal epithelium which largely requires the Jchain [30,31], Analysis of feces revealed lower IgA amounts in the fecal supernatants isolated from J-DTR mice (FIG. 12K). In total, ASC generation appeared normal in J-DTR animals excluding tissue-specific alterations in IgA levels.DT treatment leads to acute ASC depletion in J-DTR mice

[0147] To test the functionality of the J-DTR mouse model, intraperitoneal (i.p.) injections of either phosphate buffered saline (PBS) or 200 ng DT (100 pL volume) were administered to both female and male, 3-4 months old WT and J-DTR animals (FIGs. 13, 14 and FIGs. 20-21). The next day, mice were euthanized and ASC populations in the SPL, BM and THY were examined by flow cytometry (FIGs. 4A, 13B). DT had no overall impact on cellularity in any organ from either genotype ((FIGs. 13C-13E). In all 3 organs assessed, DT treatment led to significant reductions in ASC numbers only in J-DTR mice (FIGs. 13F-13H). Notably, there was some variability in this effect as SPL (FIG. 13F), BM (FIG. 13G) and THY(FIG. 13H) ASCs were reduced by ~40x, 33x and 12x, respectively. The analysis of DTR expression indicated some differences based upon maturation status (FIGs. 13F, 13H) with PCs having higher expression than PBs in the SPL and THY. As such, we also assessed if DT-mediated ablation differentially impacted PBs versus PCs (FIGs. 13I-13K). Independent of organ, DT injection led to a significant depletion of PBs and PCs in J-DTR mice with the overall magnitude (i.e., fold difference) being highest for PCs (FIGs. 13I-13K). This suggested that DT-mediated elimination of PCs was more efficient compared to PBs. To support this, we used PB and PC numbers to generate a PB:PC ratio for the SPL (FIG. 20B), BM (FIG. 20C) and THY (FIG. 20D) of J-DTR mice that received either PBS or DT. Following DT treatment of J-DTR mice, the PB:PC ratio increased suggesting a population shift to a more immature PB phenotype (FIGs. 20B-20D). Furthermore, it was observed that residual ASCs present in DT-treated J-DTR mice expressed significantly less DTR compared to PBS- treated J-DTR animals (FIGs. 20E-20G); however, the significance of this observation remains to be determined.

[0148] Phenotyping of J-DTR mice indicated that mlgA+ASCs expressed the highest levels of DTR (FIGs. 18C-18E). To determine if DT selectively ablated IgA ASCs, mlgM versus mlgA flow cytometry on ASCs from the SPL, BM and THY of J-DTR mice that received either PBS or DT was performed (FIGs. 20H-20J). DT injection led to a reduction in mlgM+, mlgA+and DN ASCs in the SPL (FIG. 20H), BM (FIG. 20I) and THY (FIG. 20J). Based upon cell numbers, it appeared that mlgA+ASCs were more efficiently depleted in the SPL (mlgM+: 30x fold decrease; mlgA+: 773x fold decrease; DN: 285x fold decrease) and BM (mlgM+: 20x fold decrease; mlgA+: 211x fold decrease; DN: 51x fold decrease) (FIGs. 20H-20I). In the THY, mlgM+(16x fold decrease) and mlgA+(12x fold decrease) ASCs were depleted at a relatively similar magnitude with THY DN ASCs being reduced by ~7x (FIG. 20J).

[0149] Depletion was previously measured based upon flow cytometry and cellular identification via cell surface markers. Thus, ELISpot assays (FIGs. 14A-14J) on the SPL, BM and THY were performed to provide functional confirmation of ASC depletion. In all 3 organs analyzed, administration of DT to J-DTR mice resulted in a significant reduction in the numbers of IgM, IgG and IgA spots per 105cells (FIGs. 14B-14J). DT treatment did not significantly impact the numbers of spots observed using cells from WT animals (FIGs. 14B- 14J). A previous study has demonstrated mouse serum Ab half-lives to range from 17-22 hours (polymeric IgA) to days depending on IgG isotype

[0032] , Based on those observations,plasma Abs were not evaluated in the mice as DT treatment lasted less than 24 hours from a practical standpoint. However, it was hypothesized that fecal IgA levels may be reduced in DT-treated J-DTR mice as the concentration of fecal IgA represents a balance between production and excretion. While injection of DT did not significantly alter fecal IgA in WT mice (FIG. 14K), J-DTR animals that received DT displayed a significant reduction in IgA (FIG. 14K) indicative of gut ASC depletion.

[0150] Finally, since it was previously observed low level DTR expression by selected upstream B cell populations, how DT treatment impacted these populations was also evaluated (FIGs 21A-21E). Overall, SPL and THY total B cells were not impacted by DT treatment (FIGs. 21 B-21C). However, J-DTR mice demonstrated a reduction in their SPL GCB numbers following administration of DT (~8.8x, FIG. 21 D) supporting the above observation of DTR expression in at least a portion of SPL GCBs. While this level of depletion was significant, it was far less than that observed for SPL ASCs (~40x, FIG. 13F) from the same animals. In contrast, DT had no impact on THY GCB-like cells (FIG. 21 E). Taken together, these data demonstrate that DT can induce acute ASC depletion in J-DTR mice with a limited impact on other mature B cells populations especially in the THY.DT treatment of J-DTR mice allows for kinetic analysis of ASC production

[0151] Understanding the kinetics of ASC formation and how newly generated ASCs compete with pre-existing cells for survival niches are important considerations in vaccine development. To determine if our J-DTR mice provide a suitable model to evaluate ASC differentiation kinetics, 3-4 months old female and male J-DTR mice were treated with either PBS or DT (200 ng) (FIG. 15A and FIG. 22A). Subsequently, animals were euthanized at days 1 , 3 and 7 post-treatment with B cell and ASC populations from SPL, BM and THY being analyzed by flow cytometry (FIG. 15A and FIG. 22A). Similar to above, DT treatment did not alter SPL, BM or THY cellularity in J-DTR mice (FIGs. 15B-15D).

[0152] Consistent with previous data (FIGs. 4A, 13B-13K), DT significantly reduced SPL ASCs in J-DTR mice at 1-day post-injection (~60x, FIG. 15E) with ASCs still being depleted ~10x at day 3 (FIG. 15E). By day 7, SPL ASC numbers resembled those in PBS- treated animals (FIG. 15E). Within the BM, ASCs were acutely depleted ~138x at day 1 and ultimately reached levels observed in control mice by day 7 (FIG. 15F). At day 3, BM ASCs appeared to still be reduced in DT-treated animals although this did not reach statisticalsignificance (FIG. 15F). Finally, administration of DT resulted in reduced THY ASC numbers at both day 1 (~28x, FIG. 15G) and day 3 (~13x, FIG. 15G). Similar to the SPL and BM, THY ASCs returned to normal levels by day 7 post-DT (FIG. 15G). Interestingly enough, ASCs from DT-treated J-DTR mice expressed lower DTR at day 1 compared to day 7 posttreatment in all 3 organs assessed (FIGs. 15H-15J). As with the acute depletion studies (FIG. 21), the effects of DT-treatment on total B cell and GCB (or GCB-like) populations in the SPL and THY of J-DTR animals were also assessed (FIGs. 22A-22E). The only notable changes were reflected in a significant reduction in J-DTR SPL GCBs at 1 day following DT administration (FIG. 22D) which was followed by a non-significant reduction at day 3. SPL GCBs were at normal levels at day 7 post-DT treatment (FIG. 22D). Collectively, these data demonstrate that ASCs are continuously replenished in the SPL, BM and THY of young mice and that the J-DTR mouse model provides a suitable platform to study ASC reconstitution kinetics.Discussion

[0153] The data presented here outline the creation and validation of a mouse model in which the endogenous Jchain locus drives DTR gene expression (J-DTR mice). As shown, ASCs from J-DTR mice express high amounts of DTR protein on the cell surface and can be acutely depleted following a single dose of DT. Furthermore, due to the short half-life of DT, it was demonstrated that these mice provide a platform to assay ASC differentiation kinetics following their initial ablation. Finally, all experiments were performed using both sexes showing that this model could be utilized to study ASCs in both females and males.

[0154] In generating a mouse strain to allow for the acute deletion of ASCs, the primate DTR coding sequence was inserted into the endogenous mouse Jchain locus while maintaining the fidelity of exons required for full-length Jchain expression. This genomic location was suitable in part due to the high expression of Jchain in ASCs relative to other immune cells (FIG. 16)

[0011] , In addition, data indicate that Jchain is highly expressed in all ASC subsets, although magnitude differences exist between Ab isotypes

[0028] , Along these lines, it was observed that the highest DTR expression (i.e. , gMFI) was by mlgA+ASCs (FIG. 18). Jchain plays a pivotal role in the production of pentameric IgM and dimeric IgA and genetic ablation of Jchain leads to significant alterations in IgA and to a lesser extent IgM [30,31 ,33-35], While there has been some phenotypic variation between previous Jchain'1' models, the most reproducible phenotypes consisted of increased circulating IgA anddecreased IgA secretion into the gut lumen. The latter of which most likely being due to an i nability of dimeric IgA to form and associate with the secretory chain [30,35] rather than a Io ss of IgA producing ASCs in the gut [31 ,34], Although no developmental defects in J-DTR A SC production in the SPL, BM or THY were observed (FIG. 12), it was noted that an ~4.5x in crease in plasma IgA (FIG. 12J) and a concomitant ~2x decrease in fecal IgA (FIG. 12K) in J-DTR animals. This result was surprising as analysis of ASC-enriched samples from J-DTR SPLs for both Jchain mRNA (FIG. 10F) and protein (FIG. 17B) displayed no obvious deficie ncies relative to WT. However, gut ASCs were not directly assayed for Jchain levels due to t echnical limitations. All that being said, the IgA phenotypes present in the J-DTR mice are quite mild compared to those observed in Jchain1' mice [31 ,34,35] possibly due to our only using heterozygotes for the J-DTR allele in this study. It will be interesting to examine J- DTR homozygous mice in future studies to determine if differences in IgA localization are further exaggerated which may indicate altered Jchain protein function because of the J-DTR allele.

[0155] The J-DTR mice appear to be at least as, or more, effective as the recently published BICREAD [7] and CD138-DTR

[0014] mouse strains. This was demonstrated by the ability to deplete ASCs within 1 day following a single injection of DT. In this report, data was presented using a Logw scale for the purpose of showing the full range of ASC depletion. 100% depletion was not reached following a single dose of DT which may be a result of the previously reported limited DT half-life

[0018] combined with the time between DT treatment and terminal harvest which was ~15-18 hours for our 1 day depletion studies. It is possible that repetitive DT injections would have better “saturated the system” allowing for more complete depletion. Regardless, ASC depletion was still highly significant and clearly surpassed in magnitude what was previously achieved with antibody-mediated targeting of ASCs

[0012] , Critically, it was observed that potent depletion of all ASC isotypes examined in the SPL, BM and THY which included those expressing IgM, IgG and IgA Abs occurred (FIG. 14 and FIG. 20). These results correlated well with previously published studies that performed Jchain-CreERT2 tdTomato fluorescent labelling of ASC subsets

[0011] . Furthermore, fecal IgA concentration was used as a proxy for gut ASCs and observed a significant reduction in all but 1 J-DTR mouse which received DT (FIG. 14K). These data support the ability of intraperitoneal administration of DT to eliminate ASCs in a wide range of tissues bolstering the technical and experimental utility of the J-DTR mouse strain.

[0156] To demonstrate the feasibility of this model in terms of studying ASC differentiation kinetics, a single dose of DT was administered to J-DTR mice and assayed ASC numbers 1-, 3- and 7-days post-treatment (FIG. 15). While these experiments were limited in power, they clearly showed the ability to observe ASC reconstitution partially in the SPL at 3 days post-DT with ASC numbers returning to normal by 7 days. Reconstitution in the BM and THY was also complete by 7 days. As B cell activation and ASC production in the THY is particularly relevant to myasthenia gravis (MG) [36,37], the ability to deplete THY ASCs in J-DTR mice and study their generation within the organ may provide a critical tool that can be used to understand MG etiology

[0038] , Furthermore, it was previously demonstrated that THY ASCs possessed major histocompatibility complex class II (MHC II) on their cell surface and transcriptionally expressed machinery required for antigen presentation

[0026] , Hence, using this model to deplete THY ASCs long-term or over discrete developmental windows may prove informative regarding their potential to regulate T cell development and selection in the THY.

[0157] An interesting observation was that J-DTR ASCs remaining 1 day following DT treatment expressed low to intermediate levels of surface DTR compared to ASCs from PBS- treated J-DTR mice (FIG. 20). While it is possible that these cells would never express high DTR levels, it is suspected that their low expression may have been a sign of overall immaturity as the ASCs present after the administration of DT had increased PB:PC ratios (FIG. 20). In alignment with this, it was observed the lowest DTR levels by ASCs from J-DTR mice immediately following DT treatment when compared to those present 7 days post-DT (FIG. 15). The J-DTR model provides a platform to deplete ASCs acutely. This is particularly relevant as DT treatment of BICREAD mice would presumably also target Prdml expressing tissue resident and / or memory T cell subsets [15-17] while CD138-DTR mice may possess experimental caveats due to potentially targeting a subset of IL-10 producing CD138+macrophages

[0039] , Administration of DT to J-DTR mice did not result in alterations in organ cellularity of the SPL, BM and THY or even total B cell populations in the SPL and THY. This was an important result and indicated that widespread leaky expression of DTR was not present. However, a reduction in J-DTR SPL GCBs was seen upon DT treatment which may have been a direct effect as low levels of surface DTR expression were detected on these cells. This observation is consistent with recent data using a Jc / ia / n-driven CreERT2 in combination with a tdTomato reporter which labelled GCBs following West Nile virusvaccination

[0040] , Another study in which a green fluorescent protein (GFP) reporter was inserted alongside CreERT2 into the endogenous Jchain locus demonstrated low level GFP expression by GCBs following sheep red blood cell immunization

[0011] although tdTomato labelling (i.e., Cre activity) following Tamoxifen induction did not appear as penetrant compared to the West Nile virus study. Multiple variables may have impacted these outcomes such as antigen type and load as well as Tamoxifen dosage. Importantly, the above referenced studies first selected for GFP+or tdTomato+cells then asked whether GCBs were contained within these populations thus never explicitly addressing what percentage of the total GCB population was fluorescently marked. In contrast to SPL GCBs, GCB-like cells in the THY did not express appreciable levels of DTR nor were they depleted by DT treatment. In this study, THY GCB-like cells were defined as expressing both CD95(Fas) and GL7. These cells possess similarities to the GL7+CD38+B cell subset previously identified in the THY

[0041] and may be more akin to an activated memory B cell phenotype

[0042] , In terms of the SPL, the off-target depletion of GCBs was ~8.8x (FIG. 21 D) whereas the specific and intended elimination of ASCs was ~40x (FIG. 13F) indicating a differential activity of DT treatment as expected. Whether the low-level elimination of GCBs is relevant to the measure of ASC differentiation kinetics following immunization will most likely be context dependent, and require the inclusion of a DT-treated J-DTR control group naive to the immunogen of interest being studied.Additional SequencesNucleotide Sequence of DTRATGAAGCTGCTGCCGTCGGTGGTGCTGAAGCTCCTTCTGGCTGCAGTTCTTTCGGCACTGGTG ACTGGCGAGAGCCTGGAGCAGCTTCGGAGAGGGCTAGCTGCTGGAACCAGCAACCCGGACC CTTCCACTGGATCTACGGACCAGCTGCTACGCCTAGGAGGCGGCCGGGACCGGAAAGTCCGT GACTTGCAAGAGGCAGATCTGGACCTTTTGAGAGTCACTTTATCCTCCAAGCCACAAGCACTG GCCACACCAAGCAAGGAGGAGCACGGGAAAAGAAAGAAGAAAGGCAAGGGACTAGGGAAGA AGAGGGACCCATGTCTTCGGAAATACAAGGACTTCTGCATCCACGGAGAATGCAAATATGTGA AGGAGCTCCGGGCTCCCTCCTGCATCTGCCACCCAGGTTACCATGGAGAGAGGTGTCATGGG CTGAGCCTCCCAGTGGAAAATCGCTTATATACCTATGACCATACAACTATCCTGGCTGTGGTG GCCGTGGTGCTGTCCTCTGTCTGTCTGCTGGTCATCGTGGGGCTTCTCATGTTTAGGTACCAT AGGAGAGGTGGTTATGATGTGGAAAACGAAGAGAAAGTGAAGTTGGGCATGACTAATTCCCAC TGA (SEQ ID NO: 1)Protein Translation of DTRMKLLPSVVLKLLLAAVLSALVTGESLEQLRRGLAAGTSNPDPSTGSTDQLLRLGGGRDRKVRDLQE ADLDLLRVTLSSKPQALATPSKEEHGKRKKKGKGLGKKRDPCLRKYKDFCIHGECKYVKELRAPSC ICHPGYHGERCHGLSLPVENRLYTYDHTTILAVVAWLSSVCLLVIVGLLMFRYHRRGGYDVENEEK VKLGMTNSH- (SEQ ID NO: 2)Primers for PCR Screening:Forward OligosSQ1 : 5’- GTC AAG TAT TCC TTG CTG TGC AGA TGA TTA GG -3’ (SEQ ID NO: 3)PNDEL1 : 5’- GGT TAC CAT GGA GAG AGG TGT -3’ (SEQ ID NO: 4)NEOGT: 5’- GTC CGT GTC GCG AAG TTC CTA TAC TTT C -3’ (SEQ ID NO: 5)Reverse OligosSQ3: 5’- AAG AAC TGC AGC CAG AAG GAG -3’ (SEQ ID NO: 6)PNDEL2: 5’- ACT TCT GGG TGC AAA TGG AGA -3’ (SEQ ID NO: 7)A2: 5’- AAG CAC AGC TCT TCA TGT CAG AGA CGG -3’ (SEQ ID NO: 8) newFLPI : 5’- ACA GAG ACA AAG ACA AGC GTT AGT AGG -3’ (SEQ ID NO: 9) newFLP2: 5’- ATT TCC CAC AAC ATT AGT CAA CTC CGT TAG G-3' (SEQ ID NO: 10)WTF: 5’- CTG CAA CTC CAG TCT TTC TAG AAG ATG -3’ (SEQ ID NO: 11)WTR: 5’- CCA GCT ACA GCC TCG ATT TGT GGT G -3' (SEQ ID NO: 12)IRES cassette from JIRE KI allele:GCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGT GCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAAC CTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAG GTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAG CCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATA GTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAG AAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTACACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACAC GATGATAATATGGCCACAGAATTCTGAGCCGCCACC (SEQ ID NO: 17)References[7] X. Liu, J. Yao, Y. Zhao, J. Wang, H. Qi, Heterogeneous plasma cells and long-lived subsets in response to immunization, autoantigen and microbiota, Nature immunology. 23 (2022) 1564-1576. https: / / doi.Org / 10.1038 / S41590-022-01345-5.[9] T. Koike, K. Fujii, K. Kometani, N.S. Butler, K. Funakoshi, S. Yari, J. Kikuta, M. Ishii, T. Kurosaki, W. Ise, Progressive differentiation toward the long-lived plasma cell compartment in the bone marrow, The Journal of experimental medicine. 220 (2023). https: / / doi.org / 10.1084 / jem.20221717.

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Claims

WHAT IS CLAIMED:

1. A cell of a genetically modified non-human animal, wherein the genome of the genetically modified non-human animal comprises an exogenous diphtheria toxin receptor (DTR) gene inserted in the endogenous Jchain gene of the non-human animal, wherein the wild-type animal is insensitive to diphtheria toxin.

2. The cell of claim 1 , wherein the animal is a mouse or a rat.

3. The cell of claim 2, wherein the animal is a mouse.

4. The cell of any one of claims 1-3, wherein the DTR gene is a human or simian DTR gene.

5. The cell of any one of claims 1-3, wherein the DTR gene is inserted into an exon of the Jchain gene.

6. The cell of claim 4, wherein the exon is exon 4.

7. The cell of claim 5, wherein the DTR gene is inserted into the 3’IITR of exon 4 of the Jchain gene.

8. The cell of any one of claims 1-6, wherein the DTR gene comprises SEQ ID NO: 1 or a functional fragment thereof, or a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to SEQ ID NO: 1 or a functional fragment thereof.

9. The cell of any one of claims 1-7, wherein the cell is an embryonic stem cell.

10. A genetically modified mouse or rat embryonic stem (ES) cell, wherein the genome of the ES cell comprises an exogenous diphtheria toxin receptor (DTR) gene inserted in the endogenous Jchain gene.

11. The cell of claim 10 wherein the DTR gene is a human or simian DTR gene.

12. The cell of claim 10 or 11 , wherein the DTR gene is inserted into an exon of the Jchain gene.

13. The cell of claim 12, wherein the exon is exon 4.

14. The cell of claim 13, wherein the DTR gene is inserted into the 3’IITR of exon 4 of the Jchain gene.

15. The cell of any one of claims 10-14, wherein the DTR gene comprises SEQ ID NO: 1 or a functional fragment thereof, or a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to SEQ ID NO: 1 or a functional fragment thereof.

16. A genetically modified non-human animal whose genome comprises a diphtheria toxin receptor (DTR) gene inserted in the Jchain gene of the non-human animal, wherein the wild-type animal is insensitive to diphtheria toxin.

17. The animal of claim 16, wherein the animal is a mouse or a rat.

18. The animal of claim 17, wherein the animal is a mouse.

19. The animal or animal cell of any one of claims 16-18, wherein the DTR gene is a human or simian DTR gene.

20. The animal of any one of claims 16-19, wherein the DTR gene is inserted into an exon of the Jchain gene.

21. The animal of claim 20, wherein the exon is exon 4.

22. The animal of claim 21 , wherein the DTR gene is inserted into the 3’IITR of exon 4 of the Jchain gene.

23. The animal of any one of claims 16-22, wherein the DTR gene comprises SEQ ID NO: 1 or a functional fragment thereof, or a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to SEQ ID NO: 1 or a functional fragment thereof.

24. The animal of any one of claims 16-23, wherein the animal expresses Jchain protein at a level of at least 50%, 60%, 70%, 80%, 90% or 95% of the level that a wild type animal expresses Jchain protein.

25. The animal of any one of claims 16-24, wherein the animal expresses DTR in antibody-secreting cells (ASCs).

26. A method of making a genetically modified non-human animal or animal cell comprising modifying a genome of a non-human animal or animal cell to comprise a DTR gene, wherein the DTR gene is inserted in the Jchain gene of the non-human animal, and generating a non-human animal or animal cell comprising the modified genome, wherein the wild-type animal is insensitive to diphtheria toxin.

27. The method of claim 26, wherein said modifying comprises introducing a nucleic acid molecule comprising a DTR nucleic acid sequence into the genome of a non-human animal embryonic stem (ES) cell, obtaining a non-human animal ES cell in which the DTR nucleic acid sequence has been inserted into an exon of the endogenous Jchain gene, and generating an animal from the obtained non-human animal ES cell.

28. The method of claim 26 or 27, wherein the animal is a mouse or a rat.

29. The method of claim 38, wherein the animal is a mouse.

30. The method of any one of claims 26-29, wherein the DTR gene is inserted into an exon of the Jchain gene.

31. The method of claim 30, wherein the exon is exon 4.

32. The method of claim 31 , wherein the DTR gene is inserted into the 3’IITR of exon 4 of the Jchain gene.

33. The method of any one of claims 26-32, wherein the DTR gene comprises SEQ ID NO: 1 or a functional fragment thereof, or a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to SEQ ID NO: 1 or a functional fragment thereof.

34. The method of any one of claims 26-33, wherein the animal expresses DTR in antibody-secreting cells (ASCs).

35. A method of depleting ASCs in a non-human animal comprising administering diphtheria toxin to the non-human animal of any one of claims 16-24.

36. The method of claim 35, comprising a single administration of diphtheria toxin.

37. A use of the genetically modified animal of any one of claims 16-24 for assessing differentiation kinetics of antibody secreting cells (ASCs).

38. A use of the genetically modified animal of any one of claims 16-24 for assessing the level of antibody secreting cells (ASCs) in a tissue or organ of interest.

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