Construction method and application of a mouse model with specific knockout of the Osgep gene in pancreatic islet β cells

Abstract

The present invention relates to the field of genetic engineering technology, and particularly to a method for constructing a mouse model with specific knockout of the Osgep gene in pancreatic islet β cells and its application. The construction method includes the following steps: 1) Determine the knockout region as the exon 2 and exon 3 regions according to the structure of the Osgep gene. The CDS sequence of the Osgep gene transcript is shown in SEQ ID NO.1. Design a gene fragment with flox sites connected at both ends and containing the sequence of the knockout region, and insert this fragment into a plasmid; 2) Design sgRNA according to the upstream sequence and downstream sequence of the knockout region; 3) Mix the sgRNA, Cas9 enzyme and plasmid, and inject them together into the cytoplasm of mouse fertilized eggs to construct edited mouse fertilized eggs and produce F0 generation mice. The present invention discovers through research that Osgep knockout mice can be used for the research of diabetes, and the Osgep gene can play a role in treating diabetic mice.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a method for constructing a mouse model with specific knockout of the Osgep gene in pancreatic islet β cells and its application. Background Art

[0002] O-sialoglycoprotein endopeptidase (Osgep) is located on mouse chromosome 14 at NC_000080.7 (51152831..51162350, complement). The full-length transcript NM_133676.2 is 1608 bp, containing 11 exons. The full-length CDS sequence encoding the protein is 1008 bp, and the encoded protein contains 335 amino acid residues. Currently, the most studied function of OSGEP is to catalyze 6 A 37 modification, and transfer the TC group of threonylcarbamoyl adenylate (TC-AMP) to the 37th adenosine of tRNANNU (N = A, T, G, C) in eukaryotic cells to complete 6 A 37 modification. The Osgep gene is highly conserved in the evolution of three major species (bacteria, archaea, and eukaryotes). As early as 2004, scientists listed OSGEP as the top of the "ten genes with unknown functions" (Nucleic Acids Res, 2004. 32(18): p. 5452-63). During protein translation, tRNA modification plays a crucial role in ensuring the accuracy and efficiency of translation, and the 37th adenosine is the most highly modified, accounting for about 70% of all modifications. Evidence shows that in yeast and Drosophila cells, the lack of Osgep expression leads to a significant decrease in protein translation fidelity, causing mis-translated proteins to accumulate in cells and inducing the unfolded protein response (UPR), triggering the endoplasmic reticulum stress pathway.

[0003] Diabetes is a metabolic disease characterized by hyperglycemia. In severe cases, patients often suffer from serious complications, including retinopathy, neuropathy, and nephropathy. With the development of society and economy and the change of people's lifestyle, the prevalence of diabetes has been continuously increasing, becoming a major public health problem threatening people's health. The absolute and / or relative deficiency of insulin is considered to be the direct cause of diabetes. Insulin is the only hormone in the body that can lower blood sugar. It is synthesized and secreted by pancreatic islet β cells to maintain the normal level of blood sugar in the body. The proinsulin peptide chain synthesizes three evolutionarily conserved disulfide bonds in the endoplasmic reticulum to complete the initial folding. Normally, about 20% of the peptide chains are mis-translated and cannot maintain the correct conformation. The cell will process this part of the misfolded protein through the UPR pathway. However, under the condition of a large increase in metabolic demand, the frequency of insulin mis-translation increases, which will further exacerbate the degree of protein misfolding, causing a huge burden on the endoplasmic reticulum. Once the balance of cell processing is broken, it will lead to pancreatic islet β cell dysfunction. There is no research on the association between Osgep and pancreatic islet β cells in the existing technology. Summary of the Invention

[0004] The object of the present invention is to provide a method for constructing a mouse model with specific knockout of Osgep gene in pancreatic islet β cells and its application in diabetes research and treatment.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] A method for constructing a mouse model with specific knockout of Osgep gene in pancreatic islet β cells, comprising the following steps:

[0007] 1) Determine the knockout region as the gene fragment of exon 2 and exon 3 according to the structure of Osgep gene. The CDS sequence of Osgep gene is shown in SEQ ID NO.1; Design a gene fragment with flox sites connected at both ends and containing the knockout region sequence according to the knockout region, and insert this fragment into the plasmid;

[0008] 2) Design sgRNA according to the upstream sequence and downstream sequence of the knockout region;

[0009] 3) After mixing sgRNA, Cas9 enzyme and plasmid, inject them into the cytoplasm of mouse fertilized eggs together to construct edited mouse fertilized eggs. After in vitro culture for 1-2 hours, transplant the edited fertilized eggs into the oviduct of pseudopregnant female mice to produce F0 generation mice;

[0010] 4) Screen flox positive F0 mice from F0 generation mice, mate the positive F0 generation mice with wild mice to obtain F1 generation mice, and obtain F1 generation flox heterozygous mice;

[0011] 5) Cross the F1 generation of flox heterozygous mice with Ins-Cre mice to obtain the F2 generation of mice, and obtain the F2 generation of Osgep heterozygous islet β cell-specific knockout mice;

[0012] 6) Mate and breed the obtained F2 generation of Osgep heterozygous specific knockout mice with the F1 generation of flox positive mice to obtain a mouse model with specific knockout of the Osgep gene in islet β cells.

[0013] In one preferred embodiment, the sequences of the sgRNAs are as shown in SEQ ID NO.4-5.

[0014] Due to the large number of optional knockout regions, in fact, multiple pairs of sgRNAs (that is, one upstream and one downstream) can be designed respectively according to different knockout regions. However, since different knockout regions will affect the feasibility of the knockout results and the expression of downstream genes in the genome, some designed sgRNAs cannot obtain ideal knockout results or viable mice. And the sgRNAs shown in SEQ ID NO.4-5 have better effects.

[0015] In one preferred embodiment, the knockout region includes exon 2 and exon 3 of the Osgep gene.

[0016] In one preferred embodiment, the knockout region includes intron 2, exon 2, intron 3 and exon 3 of the Osgep gene.

[0017] In one preferred embodiment, the knockout region includes intron 2, exon 2, intron 3, exon 3 and intron 4 of the Osgep gene.

[0018] In one preferred embodiment, the sequence of the gene fragment with flox sites connected at both ends and containing the knockout region sequence is as shown in SEQ ID NO.3.

[0019] In one preferred embodiment, after designing the gene fragment with flox sites connected at both ends and containing the knockout region sequence, this gene fragment is synthesized in vitro.

[0020] In one preferred embodiment, the plasmid is the Donor plasmid.

[0021] In one preferred embodiment, in step 3), the knockout region in the Osgep gene is replaced by using the cas9 enzyme cleavage technology to obtain mouse embryonic stem cells containing flox tags, that is, edited fertilized eggs.

[0022] Based on the same inventive concept, the present invention also claims protection for a mouse model with specific knockout of the Osgep gene in pancreatic islet β cells obtained by the described construction method.

[0023] Based on the same inventive concept, the present invention also claims protection for the application of the mouse model with specific knockout of the Osgep gene in pancreatic islet β cells. This mouse model with the knocked-out Osgep gene can be used as a model for diabetes research.

[0024] The inventors found that the mice with specific knockout of the Osgep gene in pancreatic islet β cells prepared by the present invention showed increased body weight, elevated fasting blood glucose, and impaired glucose tolerance, and can be used as a model for diabetes research.

[0025] Based on the same inventive concept, the present invention also claims protection for the application of the Osgep gene in the preparation of reagents or drugs for treating diabetes. The CDS sequence of the protein encoded by the Osgep gene is as shown in SEQ ID NO.1;

[0026] Based on the same inventive concept, the present invention also claims protection for the application of reagents for overexpressing the Osgep gene or reagents for inhibiting the degradation of the Osgep gene in the preparation of reagents or drugs for treating diabetes.

[0027] A vector that overexpresses the Osgep gene.

[0028] The construction of the vector overexpressing the Osgep gene refers to the prior art Research (Wash D C). 2022;2022:9784081 or Hum Gene Ther Methods. 2017;28(1):49 - 59.

[0029] In one preferred embodiment, the vector is obtained by inserting the CDS sequence of the Osgep gene into a plasmid.

[0030] In one preferred embodiment, the vector is a recombinant plasmid containing the sequence as shown in SEQ ID NO.1.

[0031] In one preferred embodiment, the vector is pAAV - CMV - Osgep - MCS - EF1 - GdGreen - WPRE, which is a recombinant plasmid containing the sequence as shown in SEQ ID NO.1.

[0032] Based on the same inventive concept, the present invention also claims protection for the application of the vector in the preparation of reagents or drugs for treating diabetes.

[0033] An adeno - associated virus that overexpresses the Osgep gene.

[0034] In one preferred embodiment, the adeno-associated virus is an adeno-associated virus constructed with the above vector and coated with the CDS sequence of the Osgep gene.

[0035] In one preferred embodiment, the adeno-associated virus is obtained by co-transfecting the above vector with the basic adeno-associated virus packaging plasmids pAAV-RC and pHelper into HEK293 cells.

[0036] In one preferred embodiment, the basic adeno-associated virus is AAV-PAN.

[0037] Based on the same inventive concept, the present invention also claims the application of adenovirus in the preparation of a reagent for treating diabetes.

[0038] Through years of research, the present invention has found that there is a definite association between the Osgep gene and diabetes. Mice with specific knockout of the Osgep gene in pancreatic islet β cells showed increased body weight, elevated fasting blood glucose, and impaired glucose tolerance, and can be used as a diabetes research model. While mice with up-regulated Osgep gene showed a slowdown in the body weight gain of diabetic mice induced by high-fat diet, a decrease in fasting blood glucose level, and an improvement in impaired glucose tolerance, indicating that the Osgep gene can play a role in treating diabetic mice. Brief Description of the Drawings

[0039] Figure 1 Schematic diagram of the insertion position of the flox fragment in Example 1;

[0040] Figure 2 Schematic diagram of the construction of the Osgep pancreatic islet β cell-specific knockout mouse model in Example 1;

[0041] Figure 3 For Osgep in Example 1 flox / - Genotype identification of mice, PCR identification results of the flox site upstream of exon 2;

[0042] Figure 4 For Osgep in Example 1 flox / - Genotype identification of mice, PCR identification results of the flox site downstream of exon 3;

[0043] Figure 5 For Osgep in Example 2 flox / flox Genotype identification results of mice;

[0044] Figure 6 Genotype identification results of Ins2-cre mice in Example 2;

[0045] Figure 7 Elevated body weight of Osgep pancreatic islet β cell-specific knockout mice (KO);

[0046] Figure 8 For Osgep pancreatic islet β-cell specific knockout mice (KO), fasting blood glucose increased;

[0047] Figure 9 For Osgep pancreatic islet β-cell specific knockout mice (KO), glucose tolerance was impaired;

[0048] Figure 10 For high-fat diet-induced Osgep pancreatic islet β-cell specific knockout mice (KO), the degree of body weight increase was exacerbated;

[0049] Figure 11 For high-fat diet-induced Osgep pancreatic islet β-cell specific knockout mice (KO), the degree of fasting blood glucose increase was exacerbated;

[0050] Figure 12 For high-fat diet-induced Osgep pancreatic islet β-cell specific knockout mice (KO), the degree of impaired glucose tolerance was exacerbated;

[0051] Figure 13 For overexpression of Osgep, the trend of body weight increase in diabetic mice was delayed;

[0052] Figure 14 For overexpression of Osgep, the blood glucose level in diabetic mice was decreased;

[0053] Figure 15 For overexpression of Osgep, the degree of impaired glucose tolerance in diabetic mice was alleviated. Detailed implementation manners

[0054] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods and techniques without specific conditions noted in the following embodiments are generally carried out according to the conventional conditions in the art or according to the conditions recommended by the manufacturer. All the following experiments are carried out on the premise of conforming to the ethical and moral regulations of biological experiments. Example 1

[0055] Construction of Osgep-flox mice

[0056] 1) The full length of the gene Osgep transcript NM_133676.2 involved in the present invention is 1608 bp, including 11 exons, and the CDS sequence encoding the protein is 1008 bp (shown in SEQ ID NO.1). The encoded protein NP_060277.1 contains 335 amino acid residues (shown in SEQ ID NO.2).

[0057] SEQ ID NO.1: CDS sequence in the Osgep transcript NM_133676.2

[0058] ATGCCCGCGG TGCTGGGGTT CGAAGGCAGC GCCAACAAGA TCGGCGTGGG CGTGGTCCGCGACGGCACGG TGCTGGCGAA CCCGCGGCGC ACTTACGTCA CGGCCCCGGG CACCGGATTC CTTCCAGGTGACACGGCCAG GCACCATCGA GCTGTTATCC TAGACCTACT GCAGGAGGCG CTAACAGAGG CAGGATTGACCTCCAAGGAC ATTGATTGTA TTGCTTTCAC CAAAGGTCCT GGCATGGGAT CCCCATTGGC TTCTGTAGCTGTTGTTGCCC GTACAGTGGC CCAGTTGTGG AATAAGCCTT TGCTTGGCGT GAACCACTGC ATAGGCCACATTGAAATGGG CCGTCTCATC ACTGGAGCCG TTAACCCAAC TGTCCTGTAT GTGAGCGGAG GAAATACCCAGGTGATTTCC TACTCAGAAC ATCGTTATCG CATCTTTGGA GAAACTATTG ATATCGCCGT GGGAAACTGCCTGGATCGTT TTGCTCGGGT GCTGAAGATT TCCAATGACC CCAGTCCAGG CTACAACATT GAGCAGATGGCAAAGCGAGG CAAGAAGCTA GTCGAGCTGC CATACACTGT AAAGGGGATG GATGTCTCGT TTTCAGGGATTCTGTCTTTC ATTGAGGATG CAGCGCAGCG AATGCTGGCC ACTGGAGAGT GTACTCCTGA AGACCTGTGTTTCTCCTTAC AGGAAACCGT GTTTGCAATG CTAGTGGAAA TCACGGAGCG AGCCATGGCA CACTGTGGCTCCAAGGAAGC CCTCATCGTC GGAGGAGTTG GATGTAACCT GAGGCTGCAG GAGATGATGG GGACAATGTGCCAGGAGCGG GGAGCCCAGC TCTTTGCAAC AGATGAGAGA TTCTGCGTTG ACAATGGAGC CATGATAGCCCAAGCTGGTT GGGAGATGTTTCAGGCTGGG CACAGGACTC CTCTCAAAGA TTCTGCAATT ACTCAGAGGTATAGGACAGA TGAAGTGGAA GTGACATGGA GGGACTAA

[0059] SEQ ID NO.2: Amino acid sequence of the Osgep-encoded protein NP_060277.1:

[0060] mpavlgfegs ankigvgvvr dgkvlanprr tyvtppgtgf lpgdtarhhr avildllqealtesgltsqd idciaytkgp gmgaplvsva vvartvaqlw nkplvgvnhc ighiemgrli tgatsptvlyvsggntqvia ysehryrifg etidiavgnc ldrfarvlki sndpspgyni eqmakrgkkl velpytvkgmdvsfsgilsf iedvahrmla tgectpedlc fslqetvfam lveiterama hcgsqealiv ggvgcnvrlqemmatmcqer garlfatder fcidngamia qagwemfrag hrtplsdsgv tqryrtdeve vtwrd

[0061] 2) The knockout region was determined to be intron 2-exon 2-intron 3-exon 3-intron 4 according to the structure of the Osgep gene. The knockout region is 1574 bp in total. As shown in the schematic diagram, flox fragments were inserted at both ends of the knockout sequence. In vitro synthesized fragment: flox1-intron 2-exon 2-intron 3-exon 3-intron 4-flox2. The sequence is shown in SEQ ID NO.3. The bold letters are the exon coding sequences, and the bold underlined letters are the flox sequences. And this fragment was inserted into the Donor plasmid; The above sequence design was optimized by the inventors multiple times. Then the designed sequence was commissioned to Shanghai Yuan Biotechnology Co., Ltd. for synthesis (The synthesis method refers to Mol Cell Probes. 2018;37:32-38.). Figure 1 As shown, flox fragments were inserted at both ends of the knockout sequence. In vitro synthesized fragment: flox1-intron 2-exon 2-intron 3-exon 3-intron 4-flox2. The sequence is shown in SEQ ID NO.3. The bold letters are the exon coding sequences, and the bold underlined letters are the flox sequences. And this fragment was inserted into the Donor plasmid; The above sequence design was optimized by the inventors multiple times. Then the designed sequence was commissioned to Shanghai Yuan Biotechnology Co., Ltd. for synthesis (The synthesis method refers to Mol Cell Probes. 2018;37:32-38.).

[0062] SEQ ID NO.3: flox1-intron 2-exon 2-intron 3-exon3-intron 4-flox2 (The bold letters are exon coding sequences, and the bold underlined letters are flox sequences):

[0063] GTGATTATCG AATATTTGAA AACAGCTGAA AAGTTAAAAT GAAACAAAAC AAAAAAACCCTCAGTAAGTT GATGCCTCAT AAACAGACTG AAAATCGAAC AGCCATTGTT TTCAGTTCTC ATGCATGAACCATTTCTCAG ATTGTGATGA CAACAAGTAG ACTGTACAGG AAAACTAGTC ACAGCCAGCT TAGTGGCTGGACTGAGAAGC CCCAAAGCCA GACTTTCACC AAAAACAGTC CAGTCACTGG TGGTCTGCTG CTGCTCTGACCCACTACAGC TTTCTGAATC CCAGAGAAAC CATGGCATCT GAGAAGGGTA CCCAGCAGAT CATTGAGATGCACCAAAACT GTAACTCCTG AAGTCAGCAG AAAGAGCCCT ATTCAGAGCA ATGCCTGACT GCATGTGGCATAACCAGTGC TTCAAATCTG AATGACCAAA GTGGAGCTTT GCTTTATCCA CCACATTTAC CCGACCTCTCATCAGCTGAA TCCGGGGGTA CCGCGTCGAG ATAACTTCGT ATAATGTATG CTATACGAAG TTATATGTTCTTGCCCAAGG TCAGTTGGGA GCTCTCTAGA AAAGGGTGTC TTCAGAACCT TGACAACTTC TCTCAGGGAAAACATTTCCA CTACAAGCAG TATGCAGAGA GAGCATGCTT TCCCACCGCA AGCAGTATGC AGGGAGAGCATGCTTTCCCA CAGTGAGCGG TATGCAGAGC ATGCTTTCCA AGAGTTTGTT AAAACTTTGG TTTGTTGCAGGAATAAACTC ATCTTTTTGG CTTATTTGTG TGTTTGTTAG TTTGTTTGAA ACAAGGTCAA CAAATCCCTGGCTGTCCTAC TCTGTAGACC AGGCTAACCT CAGACTCATG GAGATCCACC TGCTTCTGCC TTGCTGGGATTAAAGCTTTG CACCACCACC ACCCAACTAA CTGGTTTGTT GGCAAAGTGT GTTAAAGTGG TTTGTATTTTGATGAATAAA AATGTATTGG CACACGCCTT TAATCCCAGC ACTCAGGAGG CAGAGACAGG CGGATTTCTGAGTTTGAGGC CAGCCTGGTC TGCAAAGTGA GTTCCCGAAA GCCAGGGTTA TTCAGAGAAA CCGTGTCTCGAAAAAACAAA ACAAAACAAA AAAAATTAGT GATTTAAGGT TCATAGCCCC ATATCACAGA TCCCTTTGCACCAGCCTAGC ACCTGCCTTC TTGTAGTTCA TGTTCTATAT GCCTTTCCTT AGGATTCCTT CCAGGTGACACGGCCAGGCA CCATCGAGCT GTTATCCTAG ACCTACTGCA GGAGGCGCTA ACAGAGGCAG GATTGACCTCCAAGGACATT GATTGTATTG CTTTCACCAA AGGTAAGTCT GGGAGAGTTG TCAGCAAGGG CAGTATGTGGGTGGTGGGTA CAGACCACCA CCATTACACA TTTTCAAATC TACTTTCTCT ACATACTTTTCCTAATTTCTAGGATTCTAA CCACATTTGT ACGCTGTCAG GTCCTGGCAT GGGATCCCCA TTGGCTTCTG TAGCTGTTGTTGCCCGTACA GTGGCCCAGT TGTGGAATAA GCCTTTGCTT GGCGTGAACC ACTGCATAGG CCACATTGAAATGGGCCGTC TCATCACTGG AGCCGTTAAC CCAACTGTCC TGTATGTGAG CGGAGGAAAT ACCCAGGTACTTAAGAGGAC TCTTTGTATT CCATCTTAAT GCTAAGGTAT TCTACCTGAG TTATAGGAGC TGCAAGAGCAAAACCAGGCT CATTCAGACC TAAGATATTA TAGAGAATAC AACAGAGATA TGAAGTTGCA AGCTCTGGAGACAAATTAGA ATATTTGAAC TTTATACATA CAGCTAGAAT TTCTCATTTA AAAAGAAAAG AATGTGGGTTTCTTAACTTC AAGATAATGT CTCTCTCTTT TTACTTGTGT GGGTGTTTTG CCTGAATGCA CATAGGAGCTTCAGGAAGAC AGAAGAGGTT ATGAGATCCC TGGGAACTGG AGTTATAGAC AGTTGTGAGC CACTGTGTGGGTGCAGGGAA TCAAACCCAG GTCCTTTGGA ACAGCGGTTA GTGCATTTAA CTATTGGACC ATCTCTCCAGCCCTAGCATC GCATTGTCTG AGTAGGTG AT AACTTCGTAT AATGTATGCT ATACGAAGTT ATTCTGAGGCGGAAAGAACC AGGAGCTCGA ATTCATATTC ATTTATCCAC CCTGCTCTCC CTCTTTTCTA AAAGTCAGGGTCTCCAGATA ACCCAGCCCC AAAGTTGTGG TTCTCCTTCC TCAGCCTAAG CAACTGGGAT CACAAGCTACTGTGTGATGT GACTGTGGCA GGGACTCAAT GCAGTAGACA GCTTCCCTTG TACTATTTTG ATCTTCTGAACTCCTCCCAC GTGCTGGGAT TACAGACTTG CACCACCATG CCTGGCTCAC CTTTTTCTAT TTCTGACGGAATGTCTGTGT AGGTCACCAC AACAAACTCA GTTTTGCCTG TTTTAAATTC TTTTTTTGTT AGTTTGGGTTTGGTTTTTTT GTTTGTTTTG GTTTGGTTTT TGTTGGTTTT TTTGTTGTTG TTGTTTTTTG TTTTTGTTTTTTAGACAGGG TTTCTCTGTA

[0064] 3) Design sgRNAs respectively according to the upstream sequence and downstream sequence of the knockout region of the Osgep gene, which are used to guide the cas9 enzyme to cleave the knockout region of the Osgep gene in the genome. The sgRNAs upstream of the knockout region are respectively;

[0065] SEQ ID NO.4: TCAAGGTTCTGAAGACACGG TGG;

[0066] SEQ ID NO.5: GAGCAGGGTGGATAAATGAA GGG.

[0067] Since there are many options for the knockout region, in fact, multiple pairs of sgRNAs (i.e., one upstream and one downstream) can be designed respectively according to different knockout regions. However, since different knockout regions will affect the feasibility of the knockout result and the expression of downstream genes in the genome, some of the designed sgRNAs cannot obtain ideal knockout results or viable mice. The sgRNAs shown in SEQ ID NO.4-5 have better effects.

[0068] 4) Three healthy female C57BL / 6J mice at 4 - 6 weeks of age were taken. Pregnant mare serum gonadotropin (PMSG) was injected into the abdominal cavity of the female mice. Approximately 46 - 48 hours later, human chorionic gonadotropin (hCG) was injected. Ovulation could be induced 12 hours later. After the donor female mice were given hCG, they were caged with mature male mice. On the fourth day after mating, blastocysts in the uteri of the donor female mice were collected. Under a microscope, sgRNA, cas9 enzyme, and Donor plasmid were microinjected into the fertilized eggs of C57BL / 6J mice to construct specific mouse embryonic cells (fertilized eggs).

[0069] 5) After in vitro culture for 1 - 2 hours, the surviving fertilized eggs were transplanted into the oviducts of pseudopregnant female mice. The born mice were recorded as F0 - generation mice. The F0 - generation mice were identified by PCR technology to determine the insertion site, and flox - positive mice were screened from the offspring.

[0070] PCR process system ratio and reaction procedure (applicable to all PCR reactions in the present invention):

[0071] Table 1 PCR reaction system

[0072]

[0073] Table 2 PCR reaction running program

[0074]

[0075] PCR identification was performed on the flox site upstream of exon2. The primer sequences used were:

[0076] SEQ ID NO.6: 2658 - Osgep - wt - tF1 - GCATGTGGCATAACCAGTGCTTC;

[0077] SEQ ID NO.7: 2658 - Osgep - wt - tR1 - CTCTCCCTGCATACTGCTTGC.

[0078] The detection results were as Figure 3 shown. The results showed the genotype identification results of Osgep - flox heterozygous mice. The amplified fragment of the flox site was 288 bp (flox), and the amplified fragment of the wild type was 197 bp (WT).

[0079] PCR identification was performed on the flox site downstream of exon3. The primer sequences used were:

[0080] SEQ ID NO.8: Zmk - 2F4 - GCATCGCATTGTCTGAGTAGGTG

[0081] SEQ ID NO.9: 2658 - Osgep - F0 - 3tR1 - GCAGCCATGCTGGTCTACACAG

[0082] The detection results are as Figure 4 shown. The results show the genotype identification results of Osgep - flox heterozygous mice. The amplified fragment at the flox locus is 1166 bp (flox).

[0083] 6) Since the F0 generation mice may not necessarily have the ability to stably inherit, sub - crossing is required to obtain F1 generation mice with stable inheritance ability.

[0084] Cross the F0 generation mice with C57BL / 6J mice to obtain F1 generation mice. Use primers SEQ ID NO.6 and SEQ ID NO.7 to perform PCR identification of the flox locus on the F1 generation mice. There are two possible genotypes of mice. Judging according to the PCR results:

[0085] Wild - type: One band of 197 bp fragment is obtained by PCR.

[0086] Heterozygous: Two bands of 197 bp fragment and 288 bp fragment are obtained by PCR.

[0087] Obtain F1 generation flox - positive mice, which are F1 generation Osgep - flox heterozygous mice. Example 2

[0088] Construction of Osgep pancreatic islet β - cell specific knockout mice

[0089] Mate the F1 generation Osgep - flox heterozygous mice with pancreatic islet β - cell specific transgenic Ins2 - cre mice (purchased from Jiangsu Jicui Yakang Co., Ltd.), and Osgep flox / - ×Ins Cre heterozygotes can be obtained (the schematic diagram of the construction of the Osgep pancreatic islet β - cell specific knockout mouse model is as Figure 2 shown). Then mate it with the F1 generation Osgep - flox heterozygous mice, and pancreatic islet β - cell specific knockout Osgep flox / flox ×Ins Cre mice can be obtained, abbreviated as Osgep KO mice. Its genome contains two tags, flox and cre, which need to be identified separately.

[0090] Use primers SEQ ID NO.4 and SEQ ID NO.5 to perform PCR identification on the flox locus. The PCR system and procedure are as described above. The identification results are as Figure 5As shown, the wild-type amplification fragment is 197 bp; the homozygous amplification fragment is 288 bp; the heterozygous amplification fragments are two: 197 bp and 288 bp.

[0091] In addition, in order to obtain Osgep KO mice, the genotype of the tag cre also needs to be identified. The primer pair used is:

[0092] SEQ ID NO.10: Ins2-Pro-tF- CTCTGGCCATCTGCTGATCC;

[0093] SEQ ID NO.11: Ins2-Pro-tR-CGCCGCATAACCAGTGAAAC.

[0094] The identification results are as Figure 6 shown. The PCR product fragment amplified by Ins2-cre transgenic is 550 bp; there is no amplification fragment in the wild type. Example 3

[0095] The body weight of Osgep pancreatic islet β-cell specific knockout mice (Osgep KO) increases, and glucose metabolism is disordered.

[0096] Select littermate control mice (denoted as WT, Osgep flox / flox genotype mice) and Osgep pancreatic islet β-cell specific knockout mice (denoted as KO, Osgep flox / flox ×Ins Cre genotype mice), 6 mice in each group. Record the body weight changes of the mice weekly starting from 12 weeks of age. The results are as Figure 7 shown. Compared with the control group mice, the body weight growth rate of the KO group mice is faster. At 40 weeks, the average body weight is 49.4 g, which is 30.7% higher than that of the control group.

[0097] Further detect the fasting blood glucose level of the mice. Remove the feed at 8:00 am and fast for 6 hours. Collect fresh venous blood from the tip of the mouse's tail and use a Roche Accu-Chek Active blood glucose meter and the accompanying blood glucose test strips to detect the blood glucose value. The results are as Figure 8 shown. The fasting blood glucose level of the KO group mice is significantly increased.

[0098] The Intraperitoneal Glucose Tolerance Test (IPGTT) can examine the glucose tolerance of mice. The specific operation steps are as follows: After the mice are fasted for 16 hours without water deprivation, inject a glucose solution of 2 g / kg (glucose weight / mouse body weight) intraperitoneally, and detect the blood glucose levels at 0, 15, 30, 60, and 120 minutes after injecting glucose at the tip of the mouse's tail and draw a blood glucose change curve. The results of the IPGTT experiment are asFigure 9 As shown, the results proved that the KO mice showed impaired glucose tolerance and decreased ability of the body to dispose of and clear glucose. Example 4

[0099] The degree of blood glucose metabolism disorder was aggravated in high-fat diet-induced Osgep pancreatic islet β-cell specific knockout mice (Osgep KO)

[0100] Feeding adult mice with a high-fat diet (High-fat diets, HFD) containing 60% fat Kcal% for 16 weeks is a commonly used modeling method in the field of diabetes. We selected 6-week-old control group (Osgep flox / flox genotype mice) and Osgep pancreatic islet β-cell specific knockout mice (Osgep flox / flox ×Ins Cre genotype), 6 mice in each group, and continuously fed for 16 weeks. According to the experimental operations in Example 3, the body weight changes, fasting blood glucose, and IPGTT of the two groups of mice were detected.

[0101] The results showed that after high-fat diet induction, both the KO mice and the WT group were diabetic mice as indicated by the indicators. Comparatively, the body weight of the KO mice increased by 10 g compared with the WT group ( Figure 10 ), and the fasting blood glucose level was also significantly higher than that of the WT group ( Figure 11 ), and the degree of impaired glucose tolerance was aggravated ( Figure 12 ). Example 5

[0102] Adeno-associated virus delivery of Osgep improves high-fat diet-induced blood glucose metabolism disorder

[0103] In order to provide new treatment ideas for clinical diabetes, the present invention analyzed the possibility of Osgep gene overexpression as a drug. Shanghai Genechem Co., Ltd. was commissioned to construct and package an adeno-associated virus containing the Osgep gene. Details are as follows: Construct a recombinant plasmid pAAV-CMV-Osgep-MCS-EF1-GdGreen-WPRE containing the full-length Osgep CDS sequence (as shown in SEQ ID NO.1), co-transfect HEK293 cells with the adeno-associated virus packaging plasmids pAAV-RC and pHelper, and after culturing for 72 hours, collect the virus supernatant and prepare adeno-associated virus particles coated with the Osgep gene CDS sequence by ultracentrifugation (for the specific construction process, refer to the prior art Hum Gene Ther Methods. 2017;28(1):49-59.).

[0104] After being induced by a high-fat diet, the mice were randomly divided into two groups, with 6 mice in each group. The control group virus (denoted as CON) or the Osgep overexpression virus (denoted as OE-Osgep) was injected intraperitoneally into the mice. The body weight changes, fasting blood glucose, and IPGTT of the two groups of mice were detected according to the experimental operations in Example 3.

[0105] It was found that Osgep could significantly slow down the weight gain of mice ( Figure 13 ), reduce the fasting blood glucose level ( Figure 14 ), and improve the impaired glucose tolerance ( Figure 15 ), suggesting that overexpression of Osgep could effectively treat diabetic mice.

[0106] The above is the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and its concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the claims of the present invention.

Claims

1. Application of a mouse model with pancreatic β-cell-specific knockout of the Osgep gene in the preparation of a model for diabetes research.

2. The use according to claim 1, characterized in that: The method for constructing a mouse model for specifically knocking out the Osgep gene in pancreatic islet β cells comprises the following steps: 1) According to the structure of the Osgep gene, the knockout region is determined to be the 2nd and 3rd exon regions. The CDS sequence of the Osgep gene transcript is shown in SEQ ID NO.

1. A gene fragment with flox sites at both ends and containing the knockout region sequence is designed, and this fragment is inserted into the plasmid; 2) Design sgRNA according to the upstream and downstream sequences of the knockout region; 3) After mixing sgRNA, Cas9 enzyme and plasmid, inject them into the cytoplasm of mouse fertilized eggs to construct edited mouse fertilized eggs. After 1-2 hours of in vitro culture, the edited fertilized eggs are transplanted into the oviduct of pseudo-pregnant female mice to produce F0 generation mice; 4) Screening flox-positive F0 mice from F0 mice, mating the positive F0 mice with wild mice to obtain F1 mice, and obtaining F1 flox heterozygous mice; 5) F1 generation flox heterozygous mice were hybridized with Ins-Cre mice to obtain F2 generation mice, and F2 generation Osgep heterozygous pancreatic β cell-specific knockout mice were obtained; 6) The obtained F2 generation Osgep heterozygous specific knockout mice were further mated and bred with the F1 generation flox heterozygous mice to obtain a mouse model with pancreatic β-cell-specific knockout of the Osgep gene.

3. The use according to claim 2, characterized in that: The knockout region includes exon 2 and exon 3 of the Osgep gene.

4. The use according to claim 2, characterized in that: The sequence of the gene fragment with flox sites at both ends and containing the knockout region sequence is shown in SEQ ID NO.

3.

5. The use according to claim 2, characterized in that: The sequence of sgRNA is shown in SEQ ID NO.4-5.

6. The use according to claim 2, characterized in that: The plasmid is a Donor plasmid.

7. The use according to claim 2, characterized in that: In step 3), the knockout region in the Osgep gene is replaced using Cas9 enzyme cleavage technology to obtain mouse embryonic stem cells containing the flox tag.

8. Use of an agent for overexpressing the Osgep gene in preparing an agent for treating diabetes, characterized in that: The CDS sequence of the Osgep gene is shown in SEQ ID NO.1; the reagent for overexpressing the Osgep gene is an adeno-associated virus, and the adeno-associated virus overexpresses the Osgep gene.

9. Use of an agent for overexpressing the Osgep gene in the preparation of a drug for treating diabetes, characterized in that: The CDS sequence of the Osgep gene is shown in SEQ ID NO.1; the reagent for overexpressing the Osgep gene is an adeno-associated virus, and the adeno-associated virus overexpresses the Osgep gene.

10. Use of a carrier in preparing a reagent for treating diabetes, characterized in that: The vector overexpresses the Osgep gene.

11. Use of a carrier in preparing a drug for treating diabetes, characterized in that: The vector overexpresses the Osgep gene.

12. The use according to any one of claims 10 or 11, characterized in that: The vector is obtained by inserting the Osgep gene CDS sequence into a plasmid.

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