Construction method and application of mouse model with islet beta cell specific knockout Osge gene

Abstract

The invention relates to the technical field of gene engineering, in particular to a construction method and application of a mouse model with an islet beta cell specificity knockout Osge gene. The construction method comprises the following steps: 1) according to the structure of an Osge gene, determining that a knockout region is a second exon region and a third exon region, the CDS sequence of an Osge gene transcript is as shown in SEQ ID NO.1, designing a gene segment of which two ends are connected with flox sites and which comprises a knockout region sequence, and inserting the segment into a plasmid; 2) designing sgRNA according to the upstream sequence and the downstream sequence of the knockout region; and 3) uniformly mixing the sgRNA, Cas9 enzyme and plasmid, injecting the mixture into cytoplasm of the mouse fertilized eggs, and constructing and editing the mouse fertilized eggs to produce F0-generation mice. The research finds that the Osge knockout mouse can be used for researching diabetes, and the Osge gene can play a role in treating the diabetic mouse.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to a method for constructing a mouse model for specifically knocking out the Osgep gene in pancreatic islet β cells and an application thereof. Background Art

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

[0003] Diabetes is a metabolic disease characterized by high blood sugar. Severely developed patients are often accompanied by serious complications, including retinopathy, neuropathy and nephropathy. With the development of social economy and changes in people's lifestyles, the prevalence of diabetes continues to grow, and it has become a major public health problem that endangers people's health. Absolute and / or relative insufficiency of insulin is considered to be the direct cause of diabetes. Insulin is the only hypoglycemic hormone secreted by the body. It is synthesized and secreted by pancreatic β cells to maintain normal blood sugar levels in the body. The proinsulin peptide chain synthesizes three evolutionarily conserved disulfide bonds in the endoplasmic reticulum to complete the initial folding. Under normal circumstances, about 20% of the peptide chains are mistranslated and cannot maintain the correct conformation. The cell will process this part of the misfolded protein through the UPR pathway. However, in the case of a large increase in metabolic demand, the frequency of mistranslation of insulin increases, which will further aggravate the degree of protein misfolding and impose a huge burden on the endoplasmic reticulum. Once the balance of cell processing is broken, it will lead to pancreatic β cell dysfunction. There is no research in the prior art that associates Osgep with pancreatic β cells. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing a mouse model of pancreatic β cell-specific knockout of Osgep gene 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: The method for constructing a mouse model for specifically knocking out the Osgep gene in pancreatic β cells comprises the following steps: 1) According to the structure of the Osgep gene, the knockout region is determined to be a gene fragment of exons 2 and 3. The CDS sequence of the Osgep gene is shown in SEQ ID NO.1; according to the corresponding design of the knockout region, a gene fragment with flox sites connected at both ends and containing the knockout region sequence 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 F1 generation flox-positive mice to obtain a mouse model with pancreatic β-cell-specific knockout of the Osgep gene.

[0006] In a preferred embodiment, the sequence of sgRNA is shown as SEQ ID NO.4-5.

[0007] Since there are many options for knockout regions, multiple pairs of sgRNAs (i.e., one upstream and one downstream) can actually be designed 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 surviving mice. The sgRNA shown in SEQ ID NO.4-5 has a better effect.

[0008] In a preferred embodiment, the knockout region includes exon 2 and exon 3 of the Osgep gene.

[0009] In a preferred embodiment, the knockout region includes intron 2, exon 2, intron 3 and exon 3 of the Osgep gene.

[0010] In a preferred embodiment, the knockout region includes intron 2, exon 2, intron 3, exon 3 and intron 4 of the Osgep gene.

[0011] In a preferred embodiment, the sequence of the gene fragment having flox sites at both ends and comprising a knockout region sequence is shown as SEQ ID NO.3.

[0012] In a preferred embodiment, a gene fragment is designed with flox sites at both ends and containing a knockout region sequence, and then the gene fragment is synthesized in vitro.

[0013] In a preferred embodiment, the plasmid is a Donor plasmid.

[0014] In a preferred embodiment, in step 3), the knockout region in the Osgep gene is replaced using Cas9 enzyme cleavage technology to obtain mouse embryonic stem cells containing a flox tag, i.e., edited fertilized eggs.

[0015] Based on the same inventive concept, the present invention also claims protection for the pancreatic β cell-specific Osgep gene knockout mouse model obtained by the construction method.

[0016] Based on the same inventive concept, the present invention also claims to protect the application of the pancreatic β cell-specific Osgep gene knockout mouse model, and the Osgep gene knockout mouse model can be used as a model for diabetes research.

[0017] The inventors found that the pancreatic β cell-specific Osgep gene knockout mice prepared by the present invention showed increased body weight, increased fasting blood sugar, and impaired glucose tolerance, and can be used as a diabetes research model.

[0018] Based on the same inventive concept, the present invention also claims to protect the use of the Osgep gene in preparing a reagent or drug for treating diabetes. The CDS sequence of the protein encoded by the Osgep gene is shown in SEQ ID NO.1; Based on the same inventive concept, the present invention also claims to protect the use of an agent for overexpressing the Osgep gene or an agent for inhibiting the degradation of the Osgep gene in the preparation of an agent or a drug for treating diabetes.

[0019] A vector overexpressing the Osgep gene.

[0020] The construction of the vector for overexpressing the Osgep gene refers to the prior art Research (Wash DC). 2022;2022:9784081 or Hum Gene Ther Methods. 2017;28(1):49-59.

[0021] In a preferred embodiment, the vector is obtained by inserting the CDS sequence of the Osgep gene into a plasmid.

[0022] In a preferred embodiment, the vector is a recombinant plasmid comprising the sequence shown as SEQ ID NO.1.

[0023] In a preferred embodiment, the vector is pAAV-CMV-Osgep-MCS-EF1-GdGreen-WPRE, which is a recombinant plasmid containing the sequence shown in SEQ ID NO.1.

[0024] Based on the same inventive concept, the present invention also claims to protect the use of the carrier in preparing reagents or drugs for treating diabetes.

[0025] An adeno-associated virus, which overexpresses the Osgep gene.

[0026] In a preferred embodiment, the adeno-associated virus is an adeno-associated virus constructed by the above-mentioned vector and coated with the CDS sequence of the Osgep gene.

[0027] In a preferred embodiment, the adeno-associated virus is obtained by co-transfecting the above-mentioned vector with the basic adeno-associated virus packaging plasmid pAAV-RC and pHelper into HEK293 cells.

[0028] In a preferred embodiment, the basic adeno-associated virus is AAV-PAN.

[0029] Based on the same inventive concept, the present invention also claims to protect the use of adenovirus in preparing reagents for treating diabetes.

[0030] After years of research, the present invention has found that there is a definite correlation between the Osgep gene and diabetes. The Osgep gene knockout mice with specific pancreatic β cells showed increased body weight, increased fasting blood sugar, and impaired glucose tolerance, and can be used as a diabetes research model. The mice with upregulated Osgep gene showed slowed weight gain of diabetic mice induced by high-fat diet, reduced fasting blood sugar levels, and improved impaired glucose tolerance, indicating that the Osgep gene can play a role in treating diabetic mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the insertion position of the flox fragment in Example 1; Figure 2 This is a schematic diagram of the construction of the Osgep pancreatic β-cell specific knockout mouse model in Example 1; Figure 3 Osgep in Example 1 flox / - Mouse genotype identification, PCR identification results of the flox site upstream of exon 2; Figure 4 Osgep in Example 1 flox / - Mouse genotype identification, PCR identification results of the flox site downstream of exon 3; Figure 5 Osgep in Example 2 flox / flox Mouse genotype identification results; Figure 6 The genotype identification results of Ins2-cre mice in Example 2; Figure 7 The Osgep pancreatic β-cell-specific knockout mice (KO) showed increased body weight; Figure 8 The fasting blood glucose of Osgep pancreatic β-cell-specific knockout mice (KO) was elevated; Fig. 9 Osgep pancreatic β-cell-specific knockout mice (KO) have impaired glucose tolerance; Fig.10The high-fat diet induced aggravated weight gain in Osgep pancreatic β-cell-specific knockout mice (KO); Fig.11 The high-fat diet-induced increase in fasting blood glucose in Osgep pancreatic β-cell-specific knockout mice (KO) was aggravated; Fig.12 The high-fat diet-induced glucose tolerance impairment in Osgep pancreatic β-cell-specific knockout mice (KO) was aggravated; Fig.13 Osgep overexpression delayed the weight gain trend in diabetic mice; Fig.14 Overexpression of Osgep reduces blood glucose levels in diabetic mice; Fig.15 Osgep overexpression alleviates the degree of impaired glucose tolerance in diabetic mice. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods and techniques in the following examples that do not specify specific conditions are usually carried out according to conventional conditions in the art or according to the conditions recommended by the manufacturer. All of the following experiments were carried out under the premise of meeting the moral and ethical regulations of biological experiments. Example 1

[0033] Construction of Osgep-flox mice 1) The gene Osgep transcript NM_133676.2 involved in the present invention is 1608 bp in length, including 11 exons, of which the CDS sequence encoding the protein is 1008 bp (as shown in SEQ ID NO.1). The encoded protein NP_060277.1 contains 335 amino acid residues (as shown in SEQ ID NO.2).

[0034] SEQ ID NO.1: CDS sequence of Osgep transcript NM_133676.2: ATGCCCGCGG TGCTGGGGTT CGAAGGCAGC GCCAACAAGA TCGGCGTGGG CGTGGTCCGCGACGGCACGG TGCTGGCGAA CCCGCGGCGC ACTTACGTCA CGGCCCCGGG CACCGGATTC CTTCCAGGTGACACGGCCAG GCACCATCGA GCTGGAGGATTGGGTTG CTAACAGAGG CAGGATTGACCTCCAAGGAC ATTGATTGTA TTGCTTTCAC CAAAGGTCCT GGCATGGGAT CCCCATTGGC TTCTGTAGCTGTTGTTGCCC GTACAGTGGC CCAGTTGTGG AATAAGCCTT TGCTTGGCGT GAACCACTGC ATAGGCCACATTGAGTCGATTGCCG TTAACCCAAC TGTCCTGTAT GTGAGCGGAG GAAATACCCAGGTGATTTCC TACTCAGAAC ATCGTTATCG CATCTTTGGA GAAACTATTG ATATCGCCGT GGGAAACTGCCTGGATCGTT TTGCTCGGGT GCTGAAGATT TCCAATGACC CCAGTCCAGG AGACTGACTGAGGAGGACCGTT CAAGAAGCTA GTCGAGCTGC CATACACTGT AAAGGGGATG GATGTCTCGT TTTCAGGGATTCTGTCTTTC ATTGAGGATG CAGCGCAGCG AATGCTGGCC ACTGGAGAGT GTACTCCTGA AGACCTGTGTTTCTCCTTAC AGGAAACCGT GTTTGCAATGAAA GCAGTGAGCA GTCGGG CACTGTGGCTCCAAGGAAGC CCTCATCGTC GGAGGAGTTG GATGTAACCT GAGGCTGCAG GAGATGATGG GGACAATGTGCCAGGAGCGG GGAGCCCAGC TCTTTGCAAC AGATGAGAGA TTCTGCGTTG ACAATGGAGC CATGATAGCCCAAGCTGGGGTTGGTTTCAGGCTGGG CACAGGACTC CTCTCAAAGA TTCTGCAATT ACTCAGAGGTATAGGACAGA TGAAGTGGAA GTGACATGGA GGGACTAAA SEQ ID NO.2: Osgep encoded protein NP_060277.1 amino acid sequence: 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 2) According to the structure of Osgep gene, the knockout region is determined to be intron 2-exon 2-intron 3-exon3-intron 4. The total knockout region is 1574bp, as shown in the figure 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-exon3-intron 4-flox2, the sequence is shown in SEQ ID NO.3, the bold letters are exon coding sequences, and the bold underlined letters are flox sequences. The fragment was inserted into the Donor plasmid; the above sequence designs were all designed by the inventors after multiple optimizations, and then the designed sequences were commissioned to be synthesized by Heyuan Biotechnology (Shanghai) Co., Ltd. (the synthesis method refers to Mol Cell Probes. 2018;37:32-38.).

[0035] SEQ ID NO.3: flox1-intron 2-exon 2-intron 3-exon3-intron 4-flox2 (bold letters are exon coding sequences, bold underlined letters are flox sequences): 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 ATAACTTCGTATAATGTATGCTATACGAAGTTATATGTTCTTGCCCAAGG TCAGTTGGGA GCTCTCTAGA AAAGGGTGTC TTCAGAACCT TGACAACTTC TCTCAGGGAAAACATTTCCA CTACAAGCAG TATGCAGAGA GAGCATGCTT TCCCACCGCA AGCAGTATGC AGGGAGAGCATGCTTTCCCA CAGTGATCATGCCATGCCATGCTT AGAGTTTGTT AAAACTTTGG TTTGTTGCAGGAATAAACTC ATCTTTTTGG CTTATTTGTG TGTTTGTTAG TTTGTTTGAA ACAAGGTCAA CAAATCCCTGGCTGTCCTAC TCTGTAGACC AGGCTAACCT CAGACTCATG GAGATCCACC TGCTTCTGCCTTGCCTTGACCTTGACCTTGA ACCCAACTAA CTGGTTTGTT GGCAAAGTGT GTTAAAGTGG TTTGTATTTTGATGAATAAA AATGTATTGG CACACGCCTT TAATCCCAGC ACTCAGGAGG CAGAGACAGG CGGATTTCTGAGTTTGAGGC CAGCCTGGTC TGCAAAGTGA GTTCAGGAGAGTA GCCGAGAGATTAA CCGTGTCTCGAAAAAACAAA ACAAAACAAAA AAAAATTAGT GATTTAAGGT TCATAGCCCC ATATCCACAGA TCCCTTTGCACCAGCCTAGC ACCTGCCTTC TTGTAGTTCA TGTTCTATAT GCCTTTCCTT AGGATTCCTT CCAGGTGACACGGCCAGGCA CCATCGCTGCTGAGCCTAGCCTAGCTAGCTAGC ACAGAGGCAG GATTGACCTCCAAGGACATT GATTGTATTG CTTTCACCAA AGGTAAGTCT GGGAGAGTTG TCAGCAAGGG CAGTATGTGGGTGGTGGGTA CAGACCACCA CCATTACACA TTTTCAAATC TACTTTCTCT ACATACTTTTTCCTAATTTCTAGGATTCTAA CCACATTTGT ACGCTGTCAG GTCCTGCAT GGGATCCCCA TTGGCTTCTG TAGCTGTTGTTGCCCGTACA GTGGCCCAGT TGTGGAATAA GCCTTTGCTT GGCGTGAACC ACTGCATAGG CCACATTGAAATGGGCCGTC TCATCACTGG AGCCGTTAAC CCAACTGTCC TGTATGTGAG CGGAGGAAAT ACCCAGGTACTTAAGAGGAC TCTTTGTATT CCATCTTAAT GCTAAGGTAT TCTACCTGAG TTATAGGAGC TGCAAGAGCAAAACCAGGCT CATTCAGAC TAAGATATTA TAGAGAATAC AACAGAGATA TGAAGTTGCA AGCTCTGGGAGACAAATTAGA ATATTTGAAC TTTATACATA CAGCTAGAAT TTCTCATTTA AAAAGAAAAG AATGTGGGTTTCTAACTTC AAGATAATGT CTCTCTCTTT TTACTTGGTGT GGGTGTTTTG CCTGAATGCA CATAGGAGCTTTCAGGAAGAC 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 3) Design sgRNAs according to the upstream and downstream sequences of the knockout region of the Osgep gene, respectively, 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; SEQ ID NO.4: TCAAGGTTCTGAAGACACGG TGG; SEQ ID NO. 5: GAGCAGGTGGATAAAATGAA GGG.

[0036] Since there are many options for knockout regions, multiple pairs of sgRNAs (i.e., one upstream and one downstream) can actually be designed 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 surviving mice. The sgRNA shown in SEQ ID NO.4-5 has a better effect.

[0037] 4) Take three healthy female C57BL / 6J mice aged 4-6 weeks, inject pregnant mare serum gonadotropin (PMSG) into the female mice's peritoneal cavity, and then inject human chorionic gonadotropin (hCG) about 46-48 hours later, and ovulation can be induced 12 hours later; after giving hCG to the donor female mice, they are caged with mature male mice, and the blastocysts in the uterus of the donor female mice are collected on the fourth day after mating. Under a microscope, sgRNA, cas9 enzyme and Donor plasmid are microinjected into the fertilized eggs of C57BL / 6J mice to construct specific mouse embryonic cells (fertilized eggs); 5) After 1-2 hours of in vitro culture, the surviving fertilized eggs are transplanted into the oviduct of pseudo-pregnant mice. The mice born are recorded as F0 generation mice. The F0 generation mice are identified by PCR technology to determine the insertion site, and flox-positive mice are screened from the offspring.

[0038] PCR process system ratio and reaction flow (applicable to all PCR reactions in the present invention): Table 1 PCR reaction system

[0039] Table 2 PCR reaction program

[0040] PCR identification of the flox site upstream of exon2, the primer sequence used is: SEQ ID NO.6: 2658-Osgep-wt-tF1-GCATGTGGCATAACCAGTGCTTC; SEQ ID NO.7: 2658-Osgep-wt-tR1-CTCTCCCTGCATACTGCTTGC.

[0041] Test results such as Figure 3 The results show the genotype identification results of Osgep-flox heterozygous mice, the amplified fragment of the flox site is 288bp (flox), and the amplified fragment of the wild type is 197bp (WT).

[0042] PCR identification of the flox site downstream of exon3, the primer sequence used is: SEQ ID NO.8: Zmk-2F4-GCATCGCATTGTCTGAGTAGGTG SEQ ID NO.9: 2658-Osgep-F0-3tR1-GCAGCCATGCTGGTCTACACAG Test results such as Figure 4The results show the genotype identification results of Osgep-flox heterozygous mice, and the fragment amplified at the flox site is 1166bp (flox).

[0043] 6) Since F0 generation mice may not necessarily have the ability to inherit stably, it is necessary to pass them on to obtain F1 generation mice that can inherit stably.

[0044] F0 mice were crossed with C57BL / 6J mice to obtain F1 mice. The flox site of F1 mice was identified by PCR using primers SEQ ID NO.6 and SEQ ID NO.7. Two genotypes of mice may be produced. According to the PCR results: Wild type: PCR obtained a 197 bp fragment band.

[0045] Heterozygote: Two bands were obtained by PCR: a 197 bp fragment and a 288 bp fragment.

[0046] The F1 generation flox-positive mice were obtained, namely the F1 generation Osgep-flox heterozygous mice. Example 2

[0047] Construction of Osgep pancreatic β-cell-specific knockout mice Osgep-flox heterozygous mice of F1 generation were mated with pancreatic β-cell-specific transgenic Ins2-cre mice (purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.). flox / - ×Ins Cre Schematic diagram of the construction of heterozygous (Osgep pancreatic β-cell specific knockout mouse model Figure 2 Then, the Osgep-flox heterozygous mice of the F1 generation were mated to obtain pancreatic β-cell-specific knockout Osgep flox / flox ×Ins Cre The mouse, referred to as Osgep KO mouse, has two tags, flox and cre, in its genome, which need to be identified separately.

[0048] The flox site was identified by PCR using primers SEQ ID NO.4 and SEQ ID NO.5. The PCR system and process were as described above. Figure 5 As shown, the wild-type amplified fragment is 197 bp; the homozygous amplified fragment is 288 bp; and the heterozygous amplified fragments are two: 197 bp and 288 bp.

[0049] In addition, in order to obtain Osgep KO mice, the genotype of the tag cre needs to be identified. The primer pair used is: SEQ ID NO.10: Ins2-Pro-tF-CTCTGGCCATCTGCTGATCC; SEQ ID NO. 11: Ins2-Pro-tR-CGCCGCATAACCAGTGAAAC.

[0050] The identification results are as follows Figure 6 As shown, the PCR product fragment amplified by the Ins2-cre transgene is 550 bp; there is no amplified fragment in the wild type. Example 3

[0051] Osgep pancreatic β-cell-specific knockout mice (Osgep KO) have increased body weight and impaired blood glucose metabolism The control group of mice born in the same litter (WT, Osgep flox / flox genotype mice) and Osgep pancreatic β-cell specific knockout mice (KO, Osgep flox / flox ×Ins Cre The weight of mice was recorded weekly starting from 12 weeks of age. Figure 7 As shown, compared with the control group mice, the weight gain of the KO group mice was faster, with an average weight of 49.4g at 40 weeks, an increase of 30.7% over the control group.

[0052] To further test the fasting blood glucose level of mice, the mice were deprived of feed for 6 hours from 8:00 am, fresh venous blood was collected from the tip of the mouse tail, and the blood glucose level was tested using a Roche Active Blood Glucose Meter and matching blood glucose test strips. Figure 8 As shown, the fasting blood glucose level of KO group mice was significantly increased.

[0053] The intraperitoneal glucose tolerance test (IPGTT) can examine the glucose tolerance of mice. The specific operation steps are as follows: after the mice have fasted for 16 hours but not water, 2g / kg (glucose weight / mouse body weight) of glucose solution is injected intraperitoneally. The blood sugar level at 0, 15, 30, 60, and 120 minutes after the injection of glucose is measured at the tip of the mouse's tail and a blood sugar change curve is drawn. The results of the IPGTT experiment are as follows: Fig. 9 As shown, the results demonstrated that KO mice exhibited impaired glucose tolerance and decreased ability to dispose and clear glucose. Example 4

[0054] High-fat diet-induced hyperglycemia in Osgep pancreatic β-cell-specific knockout mice (Osgep KO) Feeding adult mice with a high-fat diet (HFD) containing 60% fat Kcal% for 16 weeks is a common modeling method in the field of diabetes. We selected a 6-week control group (Osgep flox / flox genotype mice) and Osgep pancreatic β-cell-specific knockout mice (Osgep flox / flox ×Ins Cre The two groups of mice were fed with 6 mice in each group for 16 weeks. The weight changes, fasting blood glucose and IPGTT of the two groups of mice were detected according to the experimental procedures in Example 3.

[0055] The results showed that after high-fat diet induction, the indicators of KO mice and WT group showed that both mice were diabetic. In comparison, the weight of KO mice increased by 10g ( Fig.10 ), and the fasting blood glucose level was also significantly higher than that of the WT group ( Fig.11 ), impaired glucose tolerance worsens ( Fig.12 ). Example 5

[0056] Adeno-associated virus-delivered Osgep improves high-fat diet-induced glucose metabolism disorders In order to provide a new treatment idea for clinical diabetes, the present invention analyzes whether the overexpression of the Osgep gene has the possibility of becoming a drug. Heyuan Biotechnology (Shanghai) Co., Ltd. was commissioned to construct and package an adeno-associated virus containing the Osgep gene. The details are as follows: a recombinant plasmid pAAV-CMV-Osgep-MCS-EF1-GdGreen-WPRE containing the full length of the Osgep CDS sequence (as shown in SEQ ID NO.1) was constructed, and HEK293 cells were co-transfected with the adeno-associated virus packaging plasmid pAAV-RC and pHelper. After 72 hours of culture, the viral supernatant was collected and ultracentrifuged to prepare adeno-associated virus particles coated with the Osgep gene CDS sequence (for the specific construction process, refer to the prior art Hum Gene Ther Methods. 2017;28(1):49-59.).

[0057] The mice induced by high-fat diet were randomly divided into two groups, each with 6 mice, and injected with control virus (CON) or Osgep overexpression virus (OE-Osgep) by intraperitoneal injection. The weight change, fasting blood glucose and IPGTT of the two groups of mice were detected according to the experimental procedures in Example 3.

[0058] The results showed that Osgep could significantly slow down the weight gain of mice ( Fig.13 ), lower fasting blood sugar levels ( Fig.14 ), improve impaired glucose tolerance ( Fig.15), suggesting that overexpression of Osgep can effectively treat diabetic mice.

[0059] The above is a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, can make equivalent replacements or changes according to the technical solutions and concepts of the present invention, which should be covered by the protection scope of the claims of the present invention.

Claims

1. A method for constructing a mouse model of pancreatic β-cell-specific knockout of the Osgep gene, characterized in that: The following steps are involved: 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.

2. The construction method according to claim 1, characterized in that: The knockout region includes exon 2 and exon 3 of the Osgep gene; preferably, the sequence of the gene fragment connected to flox sites at both ends and including the knockout region sequence is shown in SEQ ID NO.

3.

3. The construction method according to claim 1, characterized in that: The sequence of sgRNA is shown in SEQ ID NO.4-5.

4. The construction method according to claim 1, characterized in that: The plasmid is a Donor plasmid.

5. The construction method according to claim 1, 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.

6. A mouse model with pancreatic β cell-specific knockout of the Osgep gene obtained according to the construction method according to any one of claims 1 to 5.

7. Use of the mouse model of pancreatic β cell-specific knockout of Osgep gene according to claim 6, characterized in that: This Osgep knockout mouse model is used as a model for diabetes research.

8. Use of an agent for overexpressing the Osgep gene or an agent for inhibiting the degradation of the Osgep gene in the preparation of an agent or a drug for treating diabetes, characterized in that: The CDS sequence of the Osgep gene is shown in SEQ ID NO.

1.

9. A carrier, characterized in that The vector overexpresses the Osgep gene; preferably, the vector is obtained by inserting the CDS sequence of the Osgep gene into a plasmid.

10. Use of the carrier according to claim 9 in preparing an agent or medicine for treating diabetes.

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