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Recombinant cell with IL2RG gene and ADA gene knocked out jointly and application of recombinant cell in preparation of immunodeficient swine model

A technology of recombinant cells and immunodeficiency, applied in genetically modified cells, bone/connective tissue cells, cells modified by introducing foreign genetic material, etc., can solve problems such as damage and lymphocyte dysfunction

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

AI Technical Summary

Problems solved by technology

Deficiency of the purine nucleotidylase-related gene ADA in nucleotide metabolism-related enzymes will lead to a large enrichment of intracellular nucleotide metabolites dATP or dGTP, which have selective toxic effects on lymphocytes, resulting in lymphocyte function Impairment, impairment, or death leading to SCID

Method used

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  • Recombinant cell with IL2RG gene and ADA gene knocked out jointly and application of recombinant cell in preparation of immunodeficient swine model
  • Recombinant cell with IL2RG gene and ADA gene knocked out jointly and application of recombinant cell in preparation of immunodeficient swine model
  • Recombinant cell with IL2RG gene and ADA gene knocked out jointly and application of recombinant cell in preparation of immunodeficient swine model

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Experimental program
Comparison scheme
Effect test

Embodiment 3

[0080] 8 pigs in embodiment 3 and embodiment 4 are all just born Congjiang Xiang pigs, wherein 4 females (respectively named 1, 2, 3, 4), male 4 (named respectively A, B, C, D).

[0081]The method for preparing pig primary fibroblasts: ① Take 0.5 g of pig ear tissue, remove the hair, then soak in 75% alcohol for 30-40 seconds, and then wash with PBS buffer containing 5% (volume ratio) Penicillin-Streptomycin (Gibco) 5 times, then washed once with PBS buffer; ②cut the tissue with scissors, digest with 5mL 1% collagenase solution (Sigma) at 37℃ for 1h, then centrifuge at 500g for 5min, discard the supernatant; Resuspend the culture medium, and spread it into a cell culture dish with a diameter of 9 cm containing 10 mL of complete medium and sealed with 0.2% gelatin (VWR), and culture until the cells cover about 60% of the bottom of the dish; ④After completing step ③ , use trypsin to digest and collect the cells, and use the cell freezing medium (90% complete medium + 10% DMSO, ...

Embodiment 1

[0083] Embodiment 1, the preparation of plasmid

[0084] 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.

[0085] 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.

[0086] The plasmid pKG-U6gRNA was prepared, as shown in SEQ ID NO:3.

[0087] Plasmid pX330, plasmid pKG-GE3, and plasmid pKG-U6gRNA are all circular plasmids.

[0088] The schematic diagram of the structure of plasmid pX330 is shown in figure 1 . In SEQ ID NO: 1, the 440-725 nucleotides form the CMV enhancer, the 727-1208 nucleotides form the chickenβ-actin promoter, and the 1304-1324 nucleotides 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

[0092] Embodiment 2, the effect comparison of plasmid pX330 and plasmid pKG-GE3

[0093] Select two gRNA targets located at the MSTN gene:

[0094] Target of MSTN-gRNA1: 5'-GCTGATTGTTGCTGGTCCCG-3';

[0095] Target of MSTN-gRNA2: 5'-TTTCCAGGCGAAGTTTACTG-3'.

[0096] Select two gRNA targets located at the FNDC5 gene:

[0097] Target of FNDC5-gRNA1: 5'-TGTACTCAGTGTCCTCCTCC-3';

[0098] Target of FNDC5-gRNA2: 5'-GCTCTTCAAGACGCCTCGCG-3'.

[0099] Primers used to amplify target-containing fragments are:

[0100] MSTN-F896: 5'-TCTCTCAGACAGTGCAGGCATTA-3';

[0101] MSTN-R1351: 5'-CGTTTCCGTCGTAGCGTGATAAT-3'.

[0102] FNDC5-F209: 5'-CAGTTCTCACTTGATGGCCTTGG-3';

[0103] FNDC5-R718: 5'-AGGGGTCTGGGGAGGAATGG-3'.

[0104] 1. Preparation of recombinant plasmids

[0105] The plasmid pKG-U6gRNA was taken, digested with restriction endonuclease BbsI, and the vector backbone (a large linear fragment of about 3 kb) was recovered.

[0106] MSTN-1S and MSTN-1A were synthesized separately, t...

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Abstract

The invention discloses a recombinant cell with IL2RG gene and ADA gene knocked out jointly and application of the recombinant cell in preparation of an immunodeficient swine model. The invention provides application of an sgRNA (small guide ribonucleic acid) combination consisting of sgRNAIL2RG-g7 and sgRNAADA-g7 in preparation of a kit. The invention further provides an sgRNA (small guide ribonucleic acid) combination. The sgRNA combination is composed of sgRNAIL2RG-g7 and sgRNAADA-g7. The target sequence binding region of the sgRNAIL2RG-g7 is shown as nucleotides at the first to the twentieth site in SEQ ID NO: 12; and the target sequence binding region of the sgRNAADA-g7 is shown as nucleotides at the first to the twentieth site in SEQ ID NO: 22. The invention also discloses application of the sgRNA combination in preparation of recombinant cells and application of the sgRNA combination in preparation of an immunodeficient animal model. The invention lays a solid foundation for preparation of a severe immunodeficiency swine model, and has important application values for research and development of severe immunodeficiency medicine.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to recombinant cells knocked out of IL2RG gene and ADA gene and its application in preparing immunodeficiency pig models. Background technique [0002] Severe combined immunodeficiency (SCID) is the most severe phenotype of primary immunodeficiency disease, which refers to the simultaneous development and differentiation of T cells, B cells, and NK cells due to factors such as genetics, development, or infection. , proliferation, metabolism or dysfunction. SCID in human infants was first reported by Glanzmann and Riniker in 1950. Globally, the neonatal incidence rate of SCID is about 1 / 50000. The disease has an early age of onset, severe clinical manifestations, and high mortality. Most SCIDs are caused by abnormalities of immune-related genes, and the main inheritance modes of SCID include X-linked recessive inheritance and autosomal recessive inheritance. The onset of the disease has...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N15/62C12N15/55C12N15/12C12N5/10A01K67/027
CPCC12N15/1138C12N15/1137C12N15/8509C12N9/22C07K14/7155C12N9/78C12N5/0656A01K67/0276C12N2310/20C12Y305/04004C12N2800/107C12N2830/48C12N2830/50C07K2319/00C07K2319/09C07K2319/60C12N2510/00A01K2267/0387A01K2227/108A01K2217/075
Inventor 牛冬汪滔马翔曾为俊王磊程锐赵泽英
Owner NANJING KGENE GENETIC ENG CO LTD
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