Diabetic animal model, its construction method and application

Abstract

The present disclosure relates to a diabetic animal model and methods of constructing and using the same. In particular, the present disclosure relates to a diabetic animal model, wherein the insulin enhancer binding protein-1 (Isl-1) gene in the genome of the animal model comprises a mutation in an exon and encodes an Isl-1 polypeptide having at least one amino acid substitution compared to a wild-type Isl-1 polypeptide.

Description

Technical Field

[0001] This disclosure relates to animal models of human diseases. More specifically, this disclosure relates to non-human animal models of diabetes, methods for their construction, and their applications. Background Technology

[0002] In recent years, the incidence of early-onset type 2 diabetes has risen rapidly worldwide, with impaired insulin secretion being a characteristic feature, especially in Asia. However, the causative gene remains unclear. Genes encoding transcription factors in pancreatic β-cells are presumed to play a crucial role in the development of type 2 diabetes, as these transcription factors form a complex regulatory network in pancreatic differentiation, development, and β-cell function, involving the expression of genes related to pancreatic glucose metabolism and the insulin gene. In fact, mutations in key genes within the β-cell transcription factor regulatory network have become a cause of monogenic diabetes. For example, adolescent-onset adult diabetes (MODY) is an autosomal dominant, monogenic, early-onset type 2 diabetes.

[0003] A key driver of accurate diagnosis of diabetes is the strong influence of genetic etiology on treatment choices and clinical course. Therefore, identifying the genetic causes of autosomal dominant familial type 2 diabetes and investigating its underlying pathogenesis may contribute to understanding glycemic regulation, thereby providing new biological insights into pancreatic development, function, and insulin transcriptional regulation, as well as identifying potential new therapeutic targets for diabetes. Summary of the Invention

[0004] This disclosure covers the genetic engineering of (non-human) animals to provide animal models of diabetes. These animal models can be used to screen and develop new drugs and therapies for the prevention and / or treatment of diabetes. In some embodiments, the animal model contains mutations in the Isl-1 gene, such that the encoded Isl-1 protein has one or more point mutations.

[0005] According to one aspect of this disclosure, a diabetic animal model is provided in which the insulin enhancer-binding protein-1 (Isl-1) gene in the genome contains a mutation in an exon and encodes an Isl-1 polypeptide having at least one amino acid substitution compared to the wild-type Isl-1 polypeptide.

[0006] In some embodiments, the Isl-1 gene contains one or more point mutations in exon 5. In some embodiments, the polypeptide encoded by the Isl-1 gene has an amino acid substitution at position 283. In some embodiments, the glutamic acid (Glu, E) at position 283 of the protein encoded by the Isl-1 gene can be substituted with another amino acid residue other than E. In a specific embodiment, the glutamic acid (Glu, E) at position 283 of the polypeptide encoded by the Isl-1 gene can be substituted with aspartic acid (Asp, D), i.e., E283D. In some embodiments, the animal can express the Isl-1 polypeptide containing the E283D substitution.

[0007] In some embodiments, the animal is a non-human animal. In some embodiments, the animal can be a mammal. In some embodiments, the non-human mammal can be a primate, rabbit, goat, sheep, pig, dog, cow, or rodent. In some embodiments, the non-human animal can be a rodent, such as a rat, hamster, or mouse.

[0008] In some embodiments, the animal may be homozygous for the mutation of the Isl-1 gene. In some embodiments, the animal may be heterozygous for the mutation of the Isl-1 gene.

[0009] In some embodiments, the animal model exhibits one or more symptoms of diabetes. In some embodiments, compared to wild-type animals, the animal model exhibits symptoms of elevated blood glucose levels, decreased insulin expression levels, and / or decreased insulin secretion levels.

[0010] According to another aspect, a tissue or cell of a non-human animal derived from the animal model disclosed herein is provided.

[0011] According to another aspect, an immortalized cell is provided, said immortalized cell being prepared from cells of an animal model of the present disclosure.

[0012] According to another aspect, there is provided an embryonic stem (ES) cell of a non-human animal, the genome of which includes: one or more point mutations in the exons of the Isl-1 gene, and encodes a polypeptide with at least one amino acid substitution compared to the wild-type Isl-1 polypeptide.

[0013] In some embodiments, the Isl-1 gene contains one or more point mutations in exon 5. In some embodiments, the polypeptide encoded by the Isl-1 gene has an amino acid substitution at position 283. In some embodiments, the glutamic acid (Glu, E) at position 283 of the protein encoded by the Isl-1 gene can be substituted with another amino acid residue other than E. In a specific embodiment, the glutamic acid (Glu, E) at position 283 of the polypeptide encoded by the Isl-1 gene can be substituted with aspartic acid (Asp, D), i.e., E283D. In some embodiments, the ES cells can express the Isl-1 polypeptide containing the E283D substitution.

[0014] In some embodiments, the Isl-1 locus of the ES cells further includes one or more selection markers. In some embodiments, the selection markers may be selected from, but are not limited to, neomycin phosphotransferase (neo), hygromycin B phosphotransferase (hph), xanthine / guanine phosphotransferase (gpt), hypoxanthine phosphotransferase (Hprt), thymidine kinase (tk), and puromycin acetyltransferase (puro). In some embodiments, the inserted nucleic acid containing one or more selection markers facilitates selection of transfected ES cells. In some embodiments, the engineered Isl-1 gene further includes one or more site-specific recombinase recognition sites. In some embodiments, the engineered Isl-1 gene further includes a recombinase gene and a selection marker flanked by one or more site-specific recombinase recognition sites, said site-specific recombinase recognition sites being oriented to guide excision.

[0015] In some embodiments, one or more site-specific recombinase recognition sites include loxP, lox511, lox2272, lox2372, lox66, lox71, loxM2, lox5171, FRT, FRT11, FRT71, attp, att, FRT, Dre, rox, or combinations thereof. In some embodiments, the recombinase gene is selected from Cre, Flp (e.g., Flpe, Flpo), and Dre. In some embodiments, one or more site-specific recombinase recognition sites are lox (e.g., loxP) sites, and the recombinase gene encodes Cre recombinase.

[0016] In some implementations, the animal model does not contain selective markers.

[0017] According to another aspect of this disclosure, a non-human animal embryo is provided, which is generated from the ES cells of this disclosure.

[0018] According to another aspect of this disclosure, a method for preparing a modified non-human animal is provided, the method comprising modifying the genome of the non-human animal such that the insulin enhancer-binding protein-1 (Isl-1) gene of the modified genome contains one or more point mutations in its exons and encodes an Isl-1 polypeptide having at least one amino acid substitution compared to the wild-type Isl-1 polypeptide.

[0019] In some embodiments, the modification includes: introducing a nucleic acid sequence into the genome of an ES cell of the non-human animal to obtain a modified ES cell, wherein the insulin enhancer-binding protein-1 (Isl-1) gene of the modified ES cell contains one or more point mutations in an exon and encodes an Isl-1 polypeptide having at least one amino acid substitution compared to the wild-type Isl-1 polypeptide; and using the modified ES cell to generate the non-human animal.

[0020] In some embodiments, exon 5 of the non-human animal Isl-1 gene contains one or more mutations encoding an Isl-1 polypeptide with at least one amino acid substitution compared to the wild-type Isl-1 polypeptide. In some embodiments, exon 5 of the non-human animal Isl-1 gene contains one or more mutations encoding an Isl-1 polypeptide with an E283D amino acid substitution compared to the wild-type Isl-1 polypeptide.

[0021] In some embodiments, the step of introducing the nucleic acid sequence into the genome of the ES cells of the non-human animal includes: (1) preparing a first vector comprising a first 5' homologous arm and a first 3' homologous arm; (2) using the first vector, by homologous recombination, inserting the gene sequence of the Isl-1 gene corresponding to the space between the first 5' homologous arm and the first 3' homologous arm to obtain a second vector; and (3) by homologous recombination, replacing the fragment between the first 5' homologous arm and the first 3' homologous arm of the second vector with a fragment containing the mutation to obtain a targeting vector.

[0022] In some embodiments, the first 5' homologous arm may include a 5' nucleotide sequence homologous to the nucleotides at the Isl-1 gene locus. In some embodiments, the first 5' homologous arm may include a fragment of one of the 5' UTR, exon 1, exon 2, exon 3, exon 4, intron 1, intron 2, intron 3, and / or intron 4 of the Isl-1 gene. In some embodiments, the first 5' homologous arm may include a fragment of exon 3 of the Isl-1 gene. In some embodiments, the first 5' homologous arm may have the nucleotide sequence shown in SEQ ID NO:38 or a nucleotide sequence having at least 85% sequence identity with it.

[0023] In some embodiments, the first 3' homologous arm may include a 3' nucleotide sequence homologous to the nucleotides at the Isl-1 gene locus. In some embodiments, the first 3' homologous arm may include a fragment of intron 5, exon 6, and / or the 3' UTR of the Isl-1 gene. In some embodiments, the first 3' homologous arm may include a fragment of the 3' UTR of the Isl-1 gene. In some embodiments, the first 3' homologous arm may have the nucleotide sequence shown in SEQ ID NO: 39 or a nucleotide sequence having at least 85% sequence identity with it.

[0024] In some implementations, the segment containing the mutation in step (3) includes a second 5' homologous arm and a second 3' homologous arm.

[0025] In some embodiments, the targeting vector includes one or more selective markers between the first 5' homologous arm and the first 3' homologous arm. In some embodiments, the one or more selective markers may be located between the second 5' homologous arm and the second 3' homologous arm. In some embodiments, the segment containing the mutation in step (3) includes the second 5' homologous arm, one or more selective markers, and the second 3' homologous arm.

[0026] In some embodiments, the second 5' homologous arm may include a fragment of exon 5 and / or intron 5. In some embodiments, the second 5' homologous arm may include the mutation. In some embodiments, the second 5' homologous arm may have the nucleotide sequence shown in SEQ ID NO:40 or a nucleotide sequence having at least 85% sequence identity with it.

[0027] In some embodiments, the second 3' homologous arm may include a fragment of intron 5. In some embodiments, the second 3' homologous arm may have the nucleotide sequence shown in SEQ ID NO:41 or a nucleotide sequence having at least 85% sequence identity with it.

[0028] In some implementations, the first 5' homologous arm can be obtained by PCR using the following primer pair:

[0029] Upstream primer: 5'-AAGTGCAGCATAGGCTTCAGCAAGA-3' (SEQ ID NO:46); and

[0030] Downstream primer: 5'-CAGGCCACTTTCTGCACCACTGTGT-3' (SEQ ID NO:47).

[0031] In some implementations, the first 3' homologous arm can be obtained by PCR using the following primer pair:

[0032] Upstream primer: 5'-ATTGCAACAAGGTTACCTCTATTTT-3' (SEQ ID NO:48); and

[0033] Downstream primer: 5'-AAAAAAGACAAAACACATAAACTTA-3' (SEQ ID NO:49).

[0034] In some embodiments, the second 5' homologous arm can be obtained by PCR using the following primer pair: Isl-1-C1:

[0035] Upstream primer: 5'-ACTTGGGTATATTTACTTAGCACAT-3' (SEQ ID NO:50);

[0036] Downstream primer: 5'-ATCCCGGTACCCTCCCTCACCCCAG-3' (SEQ ID NO:51).

[0037] In some implementations, the second 3' homologous arm can be obtained by PCR using the following primer pair:

[0038] Upstream primer: 5'-AGAGAAAGCAGGATGTGGTGGTGAA-3' (SEQ ID NO:52);

[0039] Downstream primer: 5'-CACAGAACTACTTAGGCACTGAGAA-3' (SEQ ID NO:53).

[0040] In some embodiments, the animal models, cells, tissues, or ES embryonic stem cells of this disclosure can be identified using the following primer pairs:

[0041] Upstream primer: 5′-ATGCCGGGGCCGGTTCATTCAGGTT-3′ (SEQ ID NO:42); and

[0042] Downstream primer: 5′-CTGAGCCCAGAAAGCGAAGGA-3′ (SEQ ID NO:43).

[0043] In some embodiments, the animal models, cells, tissues, or ES embryonic stem cells of this disclosure can also be identified using the following primer pairs:

[0044] Upstream primer: 5′-CCTCCCCCGTGCCTTCCTTGAC-3′ (SEQ ID NO:44); and

[0045] Downstream primer: 5′-GAGTTTATGTTTGACTTGTGGGTGA-3′ (SEQ ID NO:45).

[0046] According to another aspect of this disclosure, a method for screening or identifying drugs for diabetes is provided, the method comprising administering the drug to an animal model, cell, tissue, and / or ES cell of the present disclosure. In some embodiments, the method includes performing one or more assays to determine whether the drug has a therapeutic effect on one or more symptoms of diabetes. When the drug has a therapeutic effect, the drug is identified as a therapeutic drug.

[0047] According to another aspect of this disclosure, the use of animal models, cells, tissues and / or ES cells of this disclosure in screening or identifying drugs or therapies for diabetes is provided.

[0048] In some embodiments, the diabetes may be type 1 diabetes and / or type 2 diabetes. In some embodiments, the diabetes may be adult-onset diabetes mellitus (MODY) in adolescents. Attached Figure Description

[0049] Figure 1 An example is provided of the Isl-1 gene knock-in strategy. The lengths of the recombinant homologous arms are 3.7 kb and 3.6 kb, respectively. The mutation site is in exon 5, and the mutation is located at position 849 of the coding region (G is mutated to T), corresponding to the mutation of E to D at amino acid position 283.

[0050] Figure 2 An example of a targeting vector for the Isl-1 gene is shown.

[0051] Figure 3 The PCR results of the identified homologous recombination-positive embryonic stem cell clones are shown. 5′: 5′ recombinant homologous arm; 3′: 3′ recombinant homologous arm; wt: wild-type embryonic stem cells.

[0052] Figure 4 Isl-1 is shown E283D Impaired glucose tolerance, insulin secretion, and expression in mice. (a) Weekly monitoring of body weight and (b) food intake changes in male mice. (ce) Intraperitoneal glucose tolerance test (IPGTT) was performed on HM, HE, and WT mice at 8, 12, and 16 weeks of age. (f) IPGTT assay of Isl-1. E283D Insulin secretion in mice at 16 weeks of age. Data are expressed as mean ± SEM (n = 3–6). (g) Isl-1 was measured during the intraperitoneal insulin tolerance test (IPITT). E283D Glucose levels in mice at 18 weeks of age. (h) Insulin content in the pancreatic islets of 22-week-old mice was detected by ELISA. (i) Isolated pancreatic islets of 22-week-old mice were stimulated with 2.8 and 16.7 mmol / L glucose, respectively, and incubated for 30 min. Insulin levels in the culture medium were then measured. (jk) Insulin levels in Isl-1 mice at 24 weeks of age were measured. E283D Ins2 mRNA and protein levels in mouse pancreatic islets. *p<0.05, **p<0.01, ***p<0.001, compared with WT; p<0.05, p<0.01; HE indicates heterozygote, HM indicates homozygote. AUC, area under the curve. One-way ANOVA was used to compare clinical data and laboratory parameters among the three groups.

[0053] Figure 5 Immunohistochemical staining of insulin and glucagon in pancreatic sections from 24-week-old mice is shown in (a). (bd) Quantification of islet area percentage and insulin and glucagon levels in pancreatic sections from the three genotype groups. WT, wild-type; HE, heterozygous; HM, homozygous. One-way ANOVA was used to compare clinical data and laboratory parameters among the three groups.

[0054] Figure 6 Isl-1 is shown E283D Transcriptional activity and DNA binding properties of mutants. (a)Isl-1 E283D Decreased transcriptional activity in mutants. Isl-1wt and Isl-1 E283DThe relative luciferase activity of the human insulin promoter in rat INS-1 cells was stimulated. EV: pGL3 empty vector; NC, pGL3-pcDNA3.1 negative control. (b) Western blot analysis of Isl-1WT and Isl-1 in rat INS-1 cells. E283D Protein expression. NC, pGL3-pcDNA3.1 negative control. (c)Isl-1 WT and Isl-1 E283D EMSA of mutants. Increase Isl-1WT and Isl-1 E283D The mutant was incubated with 5 moles of dsDNA in binding buffer at 25°C for 30 minutes. The hIsl-1-DNA complex and percentage are listed at the top of the gel. All experiments were repeated three times. **p<0.01, **p<0.001, compared with EV or NC; p<0.001, compared with WT. Data are expressed as mean ± SEM (n=4). One-way ANOVA was used to compare clinical data and laboratory parameters among the three groups.

[0055] Figure 7 Isl-1 is shown E283D Expression of other target genes and Isl-1 interacting proteins in mouse islets. (al) shows the mRNA levels of MafA, Pdx1, Slc2a2, glucagon, Iapp, NeuroD1, and HNF-4alpha normalized to 18S rRNA. Except for Iapp and HNF-4alpha, the levels of the other proteins were normalized to β-actin. (m,n) shows the Isl-1 expression. E283D Isl-1 mRNA and Isl-1 expression in mouse pancreatic islets. (*p<0.05, **p<0.01, vs. WT). Data are expressed as mean ± SEM of 4 mice. One-way ANOVA was used to compare clinical data and laboratory parameters among the three groups.

[0056] Figure 8 Isl-1 is shown E283D Compensatory increases in Glut2 expression were observed in mouse liver. Top panel: Quantification of Glut2 normalized to β-actin. Bottom panel: Western blot analysis of Glut2 and β-actin using protein lysates isolated from liver. HE: heterozygous; HM: homozygous. *p<0.05, **p<0.01 vs. WT. One-way ANOVA was used to compare clinical data and laboratory parameters among the three groups.

[0057] Figure 9 Isl-1 is shown WT Or Isl-1 E283DOverexpression of Isl-1 in INS1 cells. (a) Isl-1 in INS1 cells. WT and Isl-1 E283D Overexpression of [a substance] leads to the opposite change in insulin mRNA levels. (b) Isl-1 in INS1 cells WT Or Isl-1 E283D Overexpression significantly altered insulin protein expression. (cd) Insulin secreted in the supernatant and total insulin content in INS-1 cells. All experiments were repeated three times. *p<0.05, **p<0.01, ***p<0.001, vs. pcDNA3.1; p<0.05, p<0.01, p<0.001, compared with WT. Data are expressed as mean ± SEM (n=3). One-way ANOVA was used to compare clinical data and laboratory parameters among the three groups. Detailed Implementation

[0058] Isl-1 is an insulin enhancer-binding protein, a transcription factor enriched in β-cells that regulates islet cell differentiation and development, as well as the transcriptional activation of insulin. As a member of the LIM homeodomain (HD) family, Isl-1 consists of two LIM domains: an HD domain and a reactivation domain (TAD) containing 240–349 amino acids, which includes a LIM homeodomain 3 (Lhx3) binding domain (LBD) of 262–291 amino acids. Isl-1 knockout mice on islet epithelial cells died at E13.5 days due to severe hyperglycemia 3–8 weeks after birth. Furthermore, in β-cells at 8 weeks after birth, Isl-1 knockout impaired glucose tolerance and glucose-stimulated insulin secretion (GSIS) and significantly affected the β-cell transcriptome. These studies indicate that isl-1 deficiency is associated with impaired glucose tolerance or severe hyperglycemia. Furthermore, Isl-1 not only directly regulates insulin transcription and expression, but also regulates other genes such as MafA, Pdx1, Slc2a2, glucagon, and Iapp. In addition, Isl-1 interacts with two key β-cell transcription factors, NeuroD1 / Beta2 and HNF4α, synergistically enhancing insulin transcriptional activation.

[0059] Although linkage tests for Isl-1 and diabetes were negative in Nigerian, African American, and French Caucasian populations, Isl-1 showed strong linkage disequilibrium with type 2 diabetes in Asian populations (such as the Japanese population), which suggests racial heterogeneity in the inheritance of type 2 diabetes with Isl-1.

[0060] The inventors screened and identified the Isl-1 E283D mutation, which may promote the development of type 2 diabetes, by performing whole-exome sequencing (WES) on patients with a large lineage of autosomal dominant early-onset type 2 diabetes and their parents. Therefore, this disclosure provides an animal model carrying the Isl-1 E283D mutation, and characterizes the presence of the Isl-1 mutation through comprehensive in vitro and in vivo studies. E283D Phenotypic and functional characteristics of the mutant animal model. This animal model can be used to study the pathogenesis of monogenic diabetes and type 2 diabetes, and also to screen for drugs and / or treatments for monogenic diabetes and type 2 diabetes.

[0061] The residue E283 of Isl-1 is highly conserved across 99 mammalian species, and the Isl-1 protein is identical in humans, rats, and hamsters, suggesting that the E283 amino acid is highly conserved. This indicates that the protein plays a crucial role in physiological function, and that amino acid substitution at this position may be intolerable. The mouse Isl-1 protein has the following sequence: MGDMGDPPKKKRLISLCVGCGNQIHDQYILRVSPDLEWHAAACLKCAECNQYLDESCTCFVRDGKTYCKRDYIRLYGIKCAKCSIGFSKNDFVMRARSKVYHIECFRCVACSRQLIPGDEFALREDGLFCRADHDVVERASLGAGDPLSPLHPARPLQMAAEPISARQP ALRPHVHKQPEKTTRVRTVLNEKQLHTLRTCYAANPRPDALMKEQLVEMTGLSPRVIRVWFQNKRCKDKKRSIMMKQLQQQQPNDKTNIQGMTGTPMVAASPERHDGGLQANPVEVQSYQPPWKVLSDFALQSDIDQPAFQQLVNFSEGGPGSNSTGSEVASMSSQLPDTPNSMVASPIEA(SEQ ID NO:1). The Isl-1 protein mutation E283D mentioned in this article refers to the amino acid substitution at position 283 of the mouse Isl-1 protein (SEQ ID NO:1).

[0062] In some implementations, Isl-1 E283D The mouse model mimics the phenotype of type 2 diabetes, particularly MODY. In some implementations, Isl-1 E283D The mouse model exhibited glucose intolerance. In some implementations, Isl-1 E283D Mice develop diabetes around 8 weeks of age, which worsens with age. In some implementations, Isl-1 E283DMutant mice and WT mice showed significant differences in insulin sensitivity at 18 weeks of age. Notably, hyperglycemia and insulin deficiency, as detected by IPGTT in mice, and GSIS impaired in isolated islets, were more severe in homozygous (HM) mice than in heterozygous (HE) mice, as expected, suggesting this is related to Isl-1. E283D Phenotypic mutation-related dose-response. Homozygous mice showed more severe hyperglycemia and insulin deficiency as detected by IPGTT than heterozygous mice, with impaired GSIS. This suggests that in Isl-1... E283D In mouse models, Isl-1 mutations may contribute to the development of a diabetic phenotype by altering insulin secretion and sensitivity. Interestingly, these results are correlated with familial inheritance of Isl-1 mutations. E283D Patients with heterozygous mutations in diabetes have very similar phenotypes. Isl-1 directly promotes insulin transcription by binding to the insulin promoter.

[0063] In some implementations, Isl-1 E283D The mutation leads to reduced insulin transcriptional activity, thereby affecting Isl-1. E283D Insulin expression in isolated mouse pancreatic islets.

[0064] In some implementations, the islets contain Isl-1 E283D The decreased Glut2 expression induced by the mutation was associated with decreased insulin secretion and expression, which is supported by the association between impaired GSIS and significantly reduced Glut2 expression in diabetic rats. Isl-1 E283D The mutation leading to decreased Glut2 expression is due to decreased SLC2A2 transcriptional activity. In Isl-1 E283D In mice, we observed decreased Glut2 expression in the pancreas and increased Glut2 expression in the liver, consistent with the upregulation of Glut2 expression in the liver of insulin-deficient animals.

[0065] Furthermore, since Isl-1 is a positive regulator of glucagon transcription, and the E283D mutation reduces the transcriptional activity of Isl-1, Isl-1... E283D The expression of glucagon in the pancreatic islets of Langerhans in mice may be reduced. In Isl-1 E283D No significant changes in glucagon at the mRNA and protein levels were observed in the homozygous and heterozygous groups. This may be attributed to insulin's inhibition of GCG gene expression, specifically the E283D mutation significantly weakening insulin expression and secretion, thereby partially relieving insulin's inhibition of GCG gene expression.

[0066] This disclosure of Isl-1 E283D Mice exhibited impaired glucose tolerance and reduced insulin secretion. In INS-1 cells, Isl-1... E283DWhen Isl-1 is overexpressed due to mutation, insulin secretion, expression, and levels are significantly downregulated. WT Overexpression of Isl-1 significantly upregulated insulin secretion, expression, and levels. This suggests that Isl-1 may be a novel target for diabetes treatment.

[0067] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0068] definition

[0069] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0070] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0071] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0072] As used herein, the terms "targeting vector," "targeting construct," or "targeting construct" refer to a polynucleotide molecule containing a targeting region. The targeting region contains a sequence that is identical or substantially identical to a sequence in a target cell, tissue, or animal, and provides for integration of the targeting construct into a location in the genome of that cell, tissue, or animal via homologous recombination. It also includes targeting regions that are targeted using site-specific recombinase recognition sites (e.g., loxP or Frt sites). In some embodiments, the targeting constructs of this disclosure further include a nucleic acid sequence or gene of particular interest, selectable markers, control and / or regulatory sequences, and other nucleic acid sequences that allow recombination mediated by exogenous protein addition, which facilitates or contributes to recombination involving these sequences. In some embodiments, the targeting constructs of this disclosure further include a complete or partial gene of interest, wherein the gene of interest is a heterologous gene encoding a complete or partial protein having a similar function to a protein encoded by an endogenous sequence.

[0073] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are included, but those from the reference sequence are excluded.

[0074] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs, and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa. In this disclosure, when a sequence has at least 85% sequence identity with a sequence compared to that sequence, this encompasses having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.3%, at least 999.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity with that sequence.

[0075] As used herein, the term "non-human animal" refers to any non-human vertebrate. In some embodiments, non-human animals may include cyclostomes, bony fish, cartilaginous fish (e.g., sharks or rays), amphibians, reptiles, mammals, and birds. In some embodiments, the non-human animal may be a mammal. In some embodiments, the non-human mammal may be a primate, rabbit, goat, sheep, pig, dog, cow, or rodent. In some embodiments, the non-human animal may be a rodent, such as a rat, guinea pig, hamster, or mouse.

[0076] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0077] Example

[0078] method

[0079] IPGTT and IPITT detection

[0080] All mice were allowed free access to food and water and were kept in an SPF facility with a 12-hour light-dark cycle. This was observed in Isl-1 mice at 8, 12, and 16 weeks of age. E283D Intraperitoneal glucose tolerance test (IPGTT) was performed on Isl-1wt mice. After fasting overnight, glucose was injected intraperitoneally at a dose of 2 g / kg body weight. Plasma glucose and insulin levels were monitored at 0, 15, 30, 60, and 120 minutes post-injection using an automated blood glucose meter (Glucocard G+meter [GT-1820], Arkray) and a commercial ELISA kit (Abcam). Intraperitoneal insulin tolerance test (IPITT): 18-week-old mice were fasted for 6 hours and then injected intraperitoneally with insulin (0.75 U / kg human insulin, Eli Lilly, USA). Plasma glucose levels were measured at 0, 15, 30, 60, and 120 minutes post-administration. All animal experiments were reviewed and approved by the Ethics Committee of Shanghai Sixth People's Hospital, affiliated with Shanghai Jiao Tong University School of Medicine. Mice were bred and cared for according to the regulations of the Ethics Committee of Shanghai Sixth People's Hospital.

[0081] GSIS trial of islet isolation

[0082] Digestion was performed using collagenase P (COLLP-RO, Sigma) from 22-week-old Isl-1 infants that had been fasted overnight. E283D Pancreatic islets were isolated from knock-in mice. The isolated islets were pre-incubated for 1 hour in Kreb-Ringer bicarbonate-HEPES buffer containing 0.5% BSA (pH 7.4), without glucose. The pre-incubated mouse islets were then stimulated with 2.8 mM or 16.7 mM glucose at 37°C for 1 hour (10 cells per well). The supernatant was collected, and the islet cells were homogenized in lysis buffer and extracted at 4°C. After centrifugation, the insulin content and secretion of the islets were measured using ELISA.

[0083] RT-PCR, Western blot and immunohistochemistry

[0084] Total RNA was extracted from isolated pancreatic islets and detected by RT-PCR. The expression of Isl-1 target genes such as MafA, Pdx1, Slc2a2, glucagon, and Iapp, as well as interacting proteins NeuroD1 and HNF-4α, and Isl-1 itself in the islets was detected using primers listed in Table 1. Western blot analysis was performed to detect the protein expression of other Isl-1 target genes, including MafA (Santa Cruz, sc-390491), Pdx1 (Santa Cruz, sc-390792), Glut2 (NBP2-22218), glucagon (Abcam, ab92517), NeuroD1 (Santa Cruz, sc-398891), and Isl-1 itself (ThermoFisher Scientific MA5-32206), in the islets. Western blot analysis was also performed to detect Glut2 expression in the liver.

[0085] Table 1.

[0086]

[0087]

[0088] Formalin-fixed and paraffin-embedded pancreatic sections (5 μm) were dewaxed and rehydrated in graded ethanol. The sections were microwaved in 10 mmol / L citrate buffer (pH 6.0) for 5 minutes, and endogenous peroxidase was blocked with BLOXALL blocking solution (Vector Laboratories, Burlingame, CA). They were then incubated overnight at 4°C with insulin or glucagon antibodies (sc-8033 and sc-514592, Santa Cruz Biotechnology, Inc.). After washing, they were incubated with second-generation blotted HRP anti-mouse IgG at room temperature for 10 minutes. Immunostaining signals for insulin or glucagon were observed using impact DAB peroxidase (HRP) substrate. The slides were then reverse-stained with hematoxylin, dehydrated, and mounted on Vectamount (Vector Laboratories) slides.

[0089] Insulin and glucagon staining intensity (Histochemistry-Score), as well as islet area, β and α cell mass analysis were performed using Aipathwell software (Servicebio, Wuhan, China). Insulin and glucagon expression levels were determined using the H-Score method.

[0090] Luciferase reporter assay and electrophoretic mobility transfer assay (EMSA)

[0091] The insulin promoter (including the enhancing sequence, sequence number NM_000340) was synthesized and subcloned into the XhoI / HindIII site of pGL3-Basic (Promega). The pcDNA3.1 vector and pcDNA3.1-Isl-1 were then used. WT or pcDNA3.1-Isl-1 E283D The insulin promoter enhancer-luciferase construct (Shanghai Onme Biotechnology Co., Ltd.) was co-transfected into rat INS-1 cells. Transfection efficiency was normalized using the renal luciferase activity of pRL-SV40. In each experiment, co-transfection was performed (Dual-...). Reporter Assay System (Promega). Isl-1 was evaluated using Western blot analysis. WT and Isl-1 E283D Expression of the mutant. The pRL-TK plasmid (Promega) containing the Renilla luciferase gene was used as an internal control. Cells were harvested 48 hours after transfection, and luciferase activity was measured according to the supplier's instructions (Promega Inc). Each reaction was performed three times.

[0092] Purified hIsl-1 and mutant protein were incubated with 5 moles of 5′-fam-labeled dsDNA containing the synthetic insulin-enhancing sequence (see Table 2 below) at 25 °C for 30 min using 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM DTT, and 15% glycerol for EMSA. After incubation, the reaction mixture was separated on an 8% native gel to analyze the presence of the hIsl-1 and dsDNA complex. Following electrophoresis, the formation of the complex was imaged and quantified using RGB 9800 fluorescence (GE).

[0093] Table 2: Oligonucleotide sequences used in EMSA for hIsl-1 and mutant E283D.

[0094]

[0095] Note: An asterisk indicates the fluorescence group (FAM). Restriction endonuclease sites are underlined. Mutant bases are indicated by lowercase letters.

[0096] INS-1 cells were cultured at 37°C in RPMI-1640 medium containing 10% FBS, 50 μmol-β-mercaptoethanol, and 5% CO2. The empty vector pcDNA3.1 and pcDNA3.1-Isl-1 were used. WT pcDNA3.1-Isl-1 E283DRat INS-1 cells were transfected with Lipo2000 reagent for 48 h. The transfected INS-1 cells were incubated with HEPES buffer for 1 h, followed by stimulation with 16.7 mmol / L glucose for 1 h. The supernatant was collected to detect insulin secretion. Cells were harvested, and insulin expression was detected by RT-PCR and Western blotting. Insulin levels were detected using a commercial ELISA kit (Crystal Chem USA, catalog number 90080).

[0097] Statistical analysis

[0098] Clinical and laboratory data are expressed as mean ± SEM or median (interquartile range). Skewed data underwent logarithmic transformation before analysis. Clinical and laboratory parameters between groups were compared using one-way ANOVA, unpaired Student's t-test, or Pearson's χ² test. P < 0.05 was considered statistically significant. Data were analyzed and processed using SPSS 19.0 (SPSS Inc.) and GraphPad Prism 7.0 (GraphPad).

[0099] Example 1. Isl-1 E283D Construction of mouse model

[0100] 1. Construction of the targeting vector: The ISL-1 genome sequence (ENSMUSG00000042258) was obtained from the Ensembl database (http: / / www.ensembl.org / index.html), and a targeting vector was designed based on the genome sequence. The mouse ISL-1 gene has 6 exons. According to bioinformatics analysis, the point mutation location is exon 5.

[0101] The ISL-1 gene point mutation knockin vector was constructed using the ET cloning method. Figure 1 The general process is as follows: Using BAC plasmid (purchased from Source Bioscience Ltd, BAC number: BMQ-301A08) as template DNA, the four small homologous arms A, B, C, and D were amplified by PCR. The PCR primers are as follows:

[0102] Segment A:

[0103] Isl1-Aup: 5′-AAAaagcttAATGTGCAGCATAGGCTTCAGCAAGA-3′ (SEQ ID NO: 28), Isl1-Alow: 5′-GGTctcgagCAGGCCACTTTCTGCACCACTGTGT-3′ (SEQ ID NO: 29);

[0104] Segment B:

[0105] Isl1-Bup: 5′-TTActcgagATTGCAACAAGGTTACCTCTATTTT-3′ (SEQ ID NO: 30),

[0106] Isl1-Blow: 5′-TAAggatccAAAAAAGACAAACACATAAACTTA-3′ (SEQ ID NO: 31);

[0107] Segment C:

[0108] Isl1-C1-KpnI: 5′-AAGggtaccACTTGGGTATATTTACTTAGCACAT-3′ (SEQ ID NO: 32), Isl1-C4-XhoI: 5′-TTCctcgagATCCCGGTACCCTCCCTCACCCCAG-3′ (SEQ ID NO: 33);

[0109] The mutation was introduced using the following primers:

[0110] Isl1-mut-F: 5'-gtaactttgcacAtctactgggtt-3' (SEQ ID NO: 34);

[0111] Isl1-mut-R: 5'-aacccagtagaTgtgcaaagttac-3' (SEQ ID NO:35); and

[0112] Fragment D:

[0113] Isl1-Dup-BamHI: 5′-CGGggatccAGAGAAAGCAGGATGTGGTGGTGAA-3′ (SEQ ID NO: 36),

[0114] Isl1-Dlow-SacII: 5'-ACCccgcggCACAGAACTACTTAGGCACTGAGAA-3' (SEQ ID NO: 37).

[0115] The PCR system is as follows:

[0116] Template DNA (BAC plasmid): 2 μl

[0117] 10×ExTaq buffer: 10 μl

[0118] dNTP: 7 μl

[0119] Primer mixture: 4 μl

[0120] ExTaq enzyme (Takara): 1 μl,

[0121] dH2O: 76 μl.

[0122] The reaction program was: 95℃ for 5 min, 95℃ for 30 sec, 58℃ for 45 sec, 72℃ for 30 sec, 72℃ for 5 min, for 30 cycles.

[0123] The four homologous arms A, B, C, and D obtained have the following sequences:

[0124] Homologous arm A sequence:

[0125] aagtgcagcataggcttcagcaagaacgacttcgtgatgcgtgcccgctctaaggtgtaccacatcgagtgtttccgctgtgtagcctgcagccgacagctcatcccgggagacgaattcgccctgcgggaggatgggcttttctgccgtgcagaccacgatgtggtggag agagccagcctgggagctggagaccctctcagtcccttgcatccagcgcggcctctgcaaatggcagGTACTCCTCCACCCAGAGGCTGAGAAAAGGCAGGCGGGGCTAAATCATAACCTTTCTTTCCTGCAACCTAGGGGTCACACAGTGGGTGCAGAAAGTGGCCTG(SEQ ID NO:38);

[0126] Homologous arm B sequence:

[0127] attgcaacaaggttacctctattttgccacaagcgtctcgggattgtgtttgactcctgtctgtccaagaacttttcccccaaagatgtgtatagttattggttaaaatgactgttttcgctctttctggaaataaagaggaaaaaggaaactttttttgtttgctcttgcattgcaaaaattataaaagtaatttattatttattgtcaggagacttgccacttttcatgtcatttgactttttttttgtttgctgaagtaaaaagaagataaaggttgtaccgtggtctttgaattatatgtctaagtttatgtgttttgtctttttt(SEQ ID NO:39);

[0128] Homologous arm C sequence:

[0129] ACTTGGGTATATTTACTTAGCACATGCAGACCAGGAAGGGAGGGAGTTTGCTCATTAACATGTTGGGATGGGGGGGGGCGTTATTCACAGAATATCCAGGGGATGACAGGAACTCCCATGGTGGCTGCTAGTCCGGAGAGACATGATGGTGGTTTACAGGCTAACCCAGTAGATGTGCAAAGTTACCAGCCGCCCTGGAAAGTACTGAGTGACTTCGCCTTGCAAAGCGACATAGATCAGCCTGCTTTTCAGCAACTGGTAAGTGTCTGGCTCCCAAATGGGAAAAGACTGGATCCCTAACAAAAAAAGGAGACTCGTTTTAACTGTTAGGCATTGAAAGGTCCCCTGGGGTGAGGGAGGGTACCGGGAT(SEQ ID NO:40);

[0130] Homologous arm D sequence:

[0131] AGAGAAAGCAGGATGTGGTGGTGAAGAGGAGGGAGGCAGATGGAAGGAAAGGAGATCTCTTGTGAATCTTTGTTTAAAACTGAGATGCCACAATTTAACTATGTAGCCTAAGTGTGTAGTGTGATCTTCCTACAAAGGAGGCATATCTTATGGGTTGAGCTTCTCAGTGCCTAAGTAGTTCTGTG (SEQ ID NO: 41).

[0132] Arms A and B were cloned between the HindIII and BamHI restriction sites of the pBR322-2S plasmid (provided by Shanghai Southern Model Organisms) to obtain the Retrieve plasmid. Using this plasmid and BAC clones, homologous recombination was used to obtain a plasmid containing a DNA fragment with 5' and 3' homologous arms between regions A and B. Arm C of ISL-1 was cloned between the KpnI and XhoI restriction sites of the PL451 plasmid. Arm D of ISL1 was cloned between the BamHI and SacII restriction sites of the PL451 plasmid. This recombinant plasmid was then double-digested with KpnI / SacII to obtain the Neo fragment, which was then knocked into the Retrieve plasmid obtained above via homologous recombination to construct the targeting vector pBR322-MK-ISL1(KI). Figure 2 The sample was identified using restriction endonucleases and sequencing.

[0133] 2. ES cell culture: Culture dishes were coated with 0.1% Gelatin (purchased from Sigma-Aldrich) and then inoculated with mitomycin C-treated feeder cells (2 × 10⁶ cells per 10 cm dish). 6 After overnight culture, 1000 trophoblast cells can be inoculated into ES cells. The complete ES cell culture medium is DMEM (purchased from Gibco BRL) containing 1000 trophoblast cells. -6 mol / L β-mercaptoethanol, 2mM glutamine, 0.1mM non-essential amino acids, 100U / ml penicillin, 50mg / L streptomycin, 15% ES fetal bovine serum (purchased from Gibco BRL), 1000U / ml LIF (leukemia inhibitory factor, purchased from Chemicon).

[0134] 3. Electroporation DNA transfer in ES cells: ES cells in logarithmic growth phase were digested with 0.125% trypsin-EDTA and counted. An appropriate amount of PBS was added to achieve a cell density of approximately 1.5 × 10⁻⁶ cells. 7 / m1. Take 0.8ml of the above ES cell suspension, add about 35μg of NotI linearized pBR322-MK-ISL1(KI) plasmid DNA, mix well, transfer to a sterile electroporation cup and electroporate at 240V, 500μF. After resuspending, evenly distribute into three 10cm culture dishes with pre-coated feeder cells for culture.

[0135] 4. Positive and negative drug screening: After 24h and 48h of electroporation, ES cells were selectively cultured in medium containing the selection drugs G418 (final concentration of 300mg / L, purchased from Sigma-Aldrich) and Ganciclovir (final concentration of 2umol / L, purchased from Sigma-Aldrich), respectively. The culture medium was changed daily. After 7-8 days of selective culture, resistant ES cells were picked when they grew into visible clones.

[0136] 5. Selection and culture of resistant cell clones: Select resistant clones and place them in a 96-well plate (concave bottom) containing 30 μl of 0.1% trypsin-EDTA for about 3 min. Gently pipette to disperse the cells and transfer them to a 96-well culture plate for culture. After the cells have grown to 60%-80% confluence, most of the cells are frozen and stored. The remaining cells are cultured until they grow to 100% confluence and then used for genomic DNA extraction.

[0137] 6. Extraction of genomic DNA from ES cells: The culture medium was aspirated from the 96-well plate containing ES cells, and 80 μl of cell lysis buffer (containing 1 g / L proteinase K, provided by Shanghai Southern Model Biotechnology) was added to each well. After digestion at 56°C overnight, anhydrous ethanol was added to extract DNA using conventional methods, and the DNA was dissolved in 100 μl of TE.

[0138] 7. PCR identification of homologous recombination clones: such as Figure 1 As shown, ES-5-up and ES-3-low primers were designed on the outer side of the ISL-1 targeting homologous recombination arm, and ES-5-low and ES-3-up primers were designed on the neo sequence, as shown below:

[0139] ES-5-up (P1): 5′-ATGCCGGGGCCGGTTCATTCAGGTT-3′ (SEQ ID NO: 42);

[0140] ES-5-low (P2): 5′-CTGAGCCCAGAAAGCGAAGGA-3′ (SEQ ID NO: 43);

[0141] ES-3-up (P3): 5′-CCTCCCCCGTGCCTCCTTGAC-3′ (SEQ ID NO: 44);

[0142] ES-3-low (P4): 5'-GAGTTTTATGTTTGACTTGTGGGTGA-3' (SEQ ID NO: 45).

[0143] Using ES-5-up and ES-5-low primers, homologous recombination in the 5′ arm was identified. The reaction conditions were: denaturation at 95℃ for 5 min, followed by 35 cycles of 95℃ for 45 s, 64℃ for 45 s, and 72℃ for 4 min, and then 72℃ for 10 min. The homologous recombination clone should amplify a 4.5 kb fragment.

[0144] Homologous recombination in the 3′ arm was identified using ES-3-up and ES-3-low primers. The reaction conditions were: denaturation at 95℃ for 5 min, followed by 35 cycles of 94℃ for 45 s, 63℃ for 45 s, and 72℃ for 4 min, and an extension at 72℃ for 10 min. The homologous recombination clone should amplify a 4 kb fragment. The enzyme used for PCR was La Taq (TaKaRA, DRR002A), and the PCR system was described in the TaKaRA instruction manual.

[0145] Embryonic stem cell targeting was performed twice. The first targeting session had an actual voltage and discharge time of 256V and 10.4ms, yielding 159 resistant clones. The second targeting session had an actual voltage and discharge time of 256V and 10.2ms, yielding 160 resistant clones. Genomic DNA was extracted from embryonic stem cells in 96-well plates, and positive clones were further screened by PCR. The first targeting session yielded one homologous recombination clone, 1H3; the second targeting session detected seven homologous recombination clones: clones 1C7, 1C11, 1H7, 1H10, 2A6, 2F6, and 2E9 (see...). Figure 3 The PCR products of the 5′ and 3′ recombinant arms were recovered using a gel extraction kit. Enzyme digestion and sequencing confirmed that embryonic stem cell clones 1H3, 1C7, 1C11, 1H7, and 1H102A6 were correct homologous recombinant clones. Clone 1H3 was used for subsequent microinjection and blastocyst transfer.

[0146] 8. Blastocyst Injection and Embryo Transfer Using ES Cells: LIF-free DMEM complete medium was used for injection, with approximately 15 ES cells injected into each blastocyst. After injection, the blastocysts were cultured in LIF-free DMEM complete medium at 37°C and 5% CO2 for approximately 1 hour. The injected embryos were then transferred to the uterus of 2.5-day-old pseudopregnant ICR mice (purchased from Shanghai Silex Laboratory Animal Co., Ltd.), with 8-10 embryos transferred from each side. The pseudopregnant mice were housed in the SPF-grade animal facility of the Shanghai Southern Model Organisms Research Center, and gave birth naturally. The offspring were chimeric mice. A total of 9 mice were born, 6 of which were >50% chimeric males.

[0147] 9. Chimeric Mouse Breeding: Chimeric male mice were mated with C57BL / 6J purebred mice to obtain 20 gray mice derived from ES cells. Following the same ES cell identification method, positive heterozygous mice were identified using PCR. The positive mouse numbers and basic information are shown in Table 3. Seven of these mice were identified as positive heterozygous mice. The heterozygous Isl-1 mice were then... E283D Mice were hybridized to obtain homozygous Isl-1. E283D Mice.

[0148] Table 3

[0149]

[0150]

[0151] Example 2. Isl-1 food feeding E283D Diabetic phenotype in mice

[0152] Isl-1 during weeks 7-18 E283D The mice's body weight and food intake were comparable to those of the WT littermate control group. Figure 4 a, Figure 4 b). As early as 8 weeks, IPGTT observed elevated fasting plasma glucose (FPG), 2-hour postprandial glucose (2h PG), and area under the glucose curve (AUC) levels in heterozygous (HE) and homozygous (HM) mice. Glucose levels increased at 12 weeks and significantly increased at 16 weeks. Figure 4 c-4e). Furthermore, these mutant mice showed significantly reduced serum insulin secretion levels and insulin AUC at 16 weeks of age ( Figure 4 f). Furthermore, IPITT showed that, following insulin stimulation, the insulin sensitivity of mutant mice was significantly different from that of WT mice at 18 weeks of age (f). Figure 4 g).

[0153] Example 3. Isl-1 E283D Insulin secretion and expression in isolated mouse pancreatic islets

[0154] Compared with WT mice, HE and HM mice had reduced insulin levels. Figure 4 h). Compared with WT mice, 22-week-old Isl-1 E283D In mice, insulin secretion decreased at a high glucose level of 16.7 mmol / L, while there was no significant difference in insulin secretion at a low glucose level of 2.8 mmol / L. Figure 4 i). In addition, Isl-1 at 24 weeks of age E283D Decreased levels of Ins2 mRNA and protein were found in mouse pancreatic islets. Figure 4 j, Figure 4 k). Immunohistochemical staining showed Isl-1E283D Insulin levels in isolated mouse pancreatic islets were downregulated. Figure 5 a) The exocrine pancreas of the mutant mice had a similar gross morphology and histological appearance to that of the WT mice, and no signs of insulinitis were observed.

[0155] The number and size of the pancreatic islets are expressed as a percentage of the islet area. Figure 5 (b) This remained unchanged across the three groups. Compared to the WT group, the HM group showed a trend towards decreased insulin expression. Figure 5 c). There were no significant differences in glucagon levels among the three groups. Figure 5 d). In the three groups, the percentages of β cells and α cells in the total pancreatic islet cells did not change significantly.

[0156] Example 4. Isl-1 E283D Transcriptional activity of mutated insulin and EMSA

[0157] Figure 6 a shows that, compared with pcDNA3.1-mIsl-1 E283D In comparison, pcDNA3.1-mIsl-1 WT Stimulation of the human insulin promoter with the enhancer sequence-luciferase reporter gene reduced relative luciferase activity by 59.3%. Western blot results showed that transfection with pcDNA3.1-mIsl-1 WT or pcDNA3.1-mIsl-1 E283D After that, mIsl-1 WT and mIsl-1 E283D The protein expression levels are comparable. Figure 6 b). Furthermore, the E283D mutant is related to hIsl-1 WT Having almost identical insulin enhancer sequence binding ability ( Figure 6 c).

[0158] Example 5. Isl-1 E283D Expression of other target genes and Isl-1 interacting proteins in mouse pancreatic islets

[0159] We found decreased mRNA and protein expression levels of target genes MafA, Pdx1, and Slc2a2, and a slight increase in glucagon (p>0.05), while the mRNA and protein expression levels of Iapp remained unchanged. Figure 7 a- Figure 7 Furthermore, for interacting proteins, a decrease in NeuroD1 and no significant change in HNF4α mRNA were observed in the pancreatic islets. Figure 7 j- Figure 7 l). Furthermore, we also detected Isl-1 in the islets of Langerhans. E283D and Isl-1WT Expression in mouse pancreatic islets ( Figure 7 m, Figure 7 In addition, Isl-1 expression did not change significantly in the three genotype mouse groups (p > 0.05). wt and Isl-1 E283D The expression of Isl-1 in mouse islets showed no significant changes in mRNA and protein levels across the three genotype groups (WT, HE, and HM) (p>0.05).

[0160] No significant differences in glucagon mRNA and protein expression were observed in the WT, HE, and HM groups. This may be attributed to insulin inhibiting GCG gene expression, specifically the E283D mutation significantly weakening insulin expression and secretion, thereby partially relieving insulin's inhibition of GCG gene expression.

[0161] Immunohistochemical staining of glucagon showed that the glucagon content in isolated pancreatic islets of HE and HM mice was slightly upregulated compared with that in WT mice. Figure 5 a) is similar to the results of Western blot analysis. Figure 7 h).

[0162] For Isl-1 E283D Regulating the expression of other target genes in the liver revealed that, compared with WT mice, Glut2, encoded by Slc2a2, was expressed in Isl-1. E283D Significantly increased levels in the liver of mutant mice (vs. HM, p<0.05; vs. HE, p<0.01). Figure 8 ).

[0163] Example 6. Isl-1 E283D Overexpression and insulin secretion

[0164] Compared to the empty vector in transfected INS-1 cells, Isl-1 WT and Isl-1 E283D The mutants promoted the expression of insulin mRNA and protein, respectively, while Isl-1... WT In comparison, Isl-1 E283D Overexpression of [a specific substance] showed a significant inhibition of insulin expression. Figure 9 a, Figure 9 b, p < 0.05). Furthermore, overexpression of Isl-1... E283D In INS-1 cells transfected with the mutant, insulin secretion in the supernatant ( Figure 9 c, p = 0.023) and total insulin content in cells ( Figure 9 d,p=0.009) compared with Isl-1 WT reduce.

[0165] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing a diabetic mouse model, wherein, The method includes: modifying the genome of the mouse such that the modified genome binds insulin enhancer-1 (IU-1). Isl-1 The gene contains a mutation in its exons and encodes an Isl-1 polypeptide with at least one amino acid substitution compared to the wild-type Isl-1 polypeptide, the modified genome Isl-1 The gene-encoded polypeptide has an E283D amino acid substitution at position 283; The modification includes: introducing a nucleic acid sequence into the genome of the mouse's ES cells to obtain modified ES cells, wherein the modified ES cells... Isl-1 The gene contains a mutation in its exons and encodes an Isl-1 polypeptide with at least one amino acid substitution compared to the wild-type Isl-1 polypeptide; and the mice are generated using the modified ES cells. The step of introducing the nucleic acid sequence into the genome of the mouse's ES cells includes: (1) Prepare a first carrier, which includes a first 5' end homologous arm and a first 3' end homologous arm; (2) Using the first carrier, through homologous recombination, insert between the first 5' end homologous arm and the first 3' end homologous arm. Isl-1 The gene corresponds to the gene sequence between the first 5' homologous arm and the first 3' homologous arm, resulting in a second vector; and (3) By homologous recombination, the fragment between the first 5' end homologous arm and the first 3' end homologous arm of the second vector is replaced with a fragment containing the mutation to obtain a targeting vector; The first 5' homologous arm has the nucleotide sequence shown in SEQ ID NO: 38, and the first 3' homologous arm has the nucleotide sequence shown in SEQ ID NO:

39. In step (3), the mutated fragment includes a second 5' homologous arm and a second 3' homologous arm. The second 5' homologous arm has the nucleotide sequence shown in SEQ ID NO: 40, and the second 3' homologous arm has the nucleotide sequence shown in SEQ ID NO:

41.

2. The method according to claim 1, wherein, For the above Isl-1 The mutation of the gene, wherein the mouse is homozygous or heterozygous.

3. The method according to claim 1, wherein, The mouse model exhibited one or more symptoms of diabetes.

4. The method according to claim 1, wherein, Compared to wild-type animals, the mouse model exhibited symptoms of elevated blood glucose levels, decreased insulin expression levels, and / or decreased insulin secretion levels.

5. The method according to claim 1, wherein, One or more selective markers are also included between the second 5' end homologous arm and the second 3' end homologous arm.

6. A method for screening or identifying drugs for diabetes, wherein, The method includes administering a drug to a mouse model obtained by the method of any one of claims 1 to 5.

7. The method according to claim 6, wherein, The diabetes mellitus refers to type 1 diabetes and / or type 2 diabetes.

8. The method according to claim 6, wherein, The diabetes referred to is adult-onset diabetes mellitus (MODY) in adolescents.

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