CRISPR/Cas9 system and application thereof in construction of swine-derived recombinant cells with insulin receptor substrate gene defects

A technology for recombining cells and genes, applied in the direction of cells modified by introducing foreign genetic material, genetic engineering, recombinant DNA technology, etc. It can solve the problems of small number of individual samples of spontaneous disease animal models and large-scale research.

Active Publication Date: 2021-03-19
NANJING KGENE GENETIC ENG CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are pig models of type 2 diabetes induced by alloxan or streptozotocin, but experimentally induced animal disease models cannot fully sim

Method used

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  • CRISPR/Cas9 system and application thereof in construction of swine-derived recombinant cells with insulin receptor substrate gene defects
  • CRISPR/Cas9 system and application thereof in construction of swine-derived recombinant cells with insulin receptor substrate gene defects
  • CRISPR/Cas9 system and application thereof in construction of swine-derived recombinant cells with insulin receptor substrate gene defects

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Embodiment 1, the preparation of plasmid

[0100] The plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 was prepared, as shown in SEQ ID NO:1. Plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9, referred to as plasmid pX330.

[0101] The plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO was prepared, as shown in SEQ ID NO:2. Plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO, referred to as plasmid pKG-GE3.

[0102] The plasmid pKG-U6gRNA was prepared, as shown in SEQ ID NO:3.

[0103] Plasmid pX330, plasmid pKG-GE3, and plasmid pKG-U6gRNA are all circular plasmids.

[0104] The schematic diagram of the structure of plasmid pX330 is shown in figure 1 . In SEQ ID NO: 1, nucleotides 440-725 constitute the CMV enhancer, nucleotides 727-1208 constitute the chickenβ-actin promoter, and nucleotides 1304-1324 encode the SV40 nuclear localization signal (NLS ), the 1325-5449th nucleotide encodes the Cas9 protein, and the 5450-5497th nucleotide encodes the nucleoplasmin nuclear localization signal (NLS...

Embodiment 2

[0108] Example 2, Screening of Target Combinations for IRS1 Gene Knockout

[0109] 1. IRS1 gene knockout predetermined target and adjacent genome sequence conservation analysis

[0110] Pig IRS1 gene information: codes insulin receptor substrate 1; located on chromosome 15; GeneID is 100512686, Sus scrofa. The protein encoded by the porcine IRS1 gene is shown in SEQ ID NO:4. In the genomic DNA, the porcine IRS1 gene has two exons, the first exon is shown in SEQ ID NO: 6, and the second exon is shown in SEQ ID NO: 7. In the genomic DNA, part of the nucleotides upstream of the first exon of the porcine IRS1 gene is shown in SEQ ID NO:5. The open reading frame of the porcine IRS1 gene is located in the first exon, as shown in nucleotides 1-3726 in SEQ ID NO:6.

[0111] Respectively using the genomic DNA of 8 pigs as a template, using the primer pair composed of primers IRS1-GT-F412 / IRS1-GT-R1220 to carry out PCR amplification, and then perform electrophoresis, see Figure 5 ....

Embodiment 3

[0153] Example 3, Screening of Target Combinations for IRS2 Gene Knockout

[0154] 1. Conservative analysis of exons and adjacent genome sequences of IRS2 gene knockout preset targets

[0155] Pig IRS2 gene information: encoding insulin receptor substrate 2; located on chromosome 11; GeneID is 110255858, Sus scrofa. The protein encoded by the porcine IRS2 gene is shown in SEQ ID NO:11. In genomic DNA, the pig IRS2 gene has 2 exons, the coding region of the first exon is shown in the 1st-4006 nucleotides in SEQ ID NO: 12, and the coding region of the second exon As shown in nucleotides 4007-4011 in SEQ ID NO:12.

[0156] Using the genomic DNA of 8 pigs as a template, PCR amplification was carried out using a primer pair consisting of primers IRS2-GT-nF848 / IRS2-GT-nR1710, followed by electrophoresis, see Figure 8 . The PCR amplification products were recovered and sequenced, and the sequencing results were compared with the IRS2 gene sequence in the public database. Accord...

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Abstract

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system and application thereof to construction of swine-derived recombinant cells with insulin receptor substrate gene defects. The invention provides an sgRNA (ribonucleic acid) combination which is composed of sgRNAIRS1-1, sgRNAIRS1-3, sgRNAIRS2-2 and sgRNAIRS2-3. The invention provides a plasmid combination. The plasmid combination is composed of four plasmids, and the four plasmids are respectively subjected to transcription to obtain sgRNAIRS1-1, sgRNAIRS1-3, sgRNAIRS2-2 and sgRNAIRS2-3. Theapplication of the sgRNA combination or the plasmid combination is as follows: preparation of recombinant cells; preparation of a diabetic cell model; preparation of a diabetic animal model. Accordingto the invention, sgRNAIRS1-1 is as shown in SEQ ID NO:8; sgRNAIRS1-3 is as shown in SEQ ID NO:10; sgRNAIRS2-2 is shown as SEQ ID NO:14; and sgRNAIRS2-3 is as shown in SEQ ID NO:15. A solid foundation is laid for the preparation of diabetic pig models, and important application value for the research and development of diabetic medicaments is achieved.

Description

technical field [0001] The present invention relates to a CRISPR / Cas9 system and its application in the construction of pig-derived recombinant cells defective in insulin receptor substrate genes. Background technique [0002] Diabetes mellitus (DM) is a metabolic disease characterized by long-term blood sugar levels higher than the standard value. Diabetes can be divided into type 1 diabetes and type 2 diabetes, and type 2 diabetes patients account for more than 90% of the total diabetes patients. Type 1 diabetes, also known as insulin-dependent diabetes, is usually caused by the inability of the patient to secrete insulin due to the damage of pancreatic β-cells. Symptoms; type 2 diabetes, also known as non-insulin-dependent diabetes, is usually caused by the decline in the ability of insulin to regulate glucose metabolism and the decrease in insulin secretion caused by the dysfunction of pancreatic β cells. Its main clinical manifestations are obesity, fatigue, and If no...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N15/90C12N5/10A01K67/027
CPCA01K67/0276A01K2207/15A01K2217/075A01K2227/108A01K2267/0362C12N15/113C12N15/8509C12N15/907C12N2310/20
Inventor 牛冬汪滔王德华王磊程锐曾为俊马翔
Owner NANJING KGENE GENETIC ENG CO LTD
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