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Method and special kit for preparing severe immunodeficiency pig-derived recombinant cells jointly knocked out of four genes of addi

A technology of recombinant cells and immunodeficiency, applied in the direction of genetically modified cells, bone/connective tissue cells, biochemical equipment and methods, etc., can solve the problems of immune rejection, inability to carry out animal experiments, etc. The effect of low cost and high feeding cost

Active Publication Date: 2022-04-05
NANJING KGENE GENETIC ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, when studying the effect of biologically active macromolecules or cell therapy, experiments with heterologous animals will cause immune rejection, making it impossible to conduct effective animal experiments

Method used

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  • Method and special kit for preparing severe immunodeficiency pig-derived recombinant cells jointly knocked out of four genes of addi
  • Method and special kit for preparing severe immunodeficiency pig-derived recombinant cells jointly knocked out of four genes of addi
  • Method and special kit for preparing severe immunodeficiency pig-derived recombinant cells jointly knocked out of four genes of addi

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Embodiment 1, the preparation of plasmid

[0106] 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.

[0107] 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.

[0108] The plasmid pKG-U6gRNA was prepared, as shown in SEQ ID NO:3.

[0109] Plasmid pX330, plasmid pKG-GE3, and plasmid pKG-U6gRNA are all circular plasmids.

[0110] 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

[0114] Embodiment 2, the effect comparison of plasmid pX330 and plasmid pKG-GE3

[0115] Select two gRNA targets located at the MSTN gene:

[0116] Target of MSTN-gRNA1: 5'-GCTGATTGTTGCTGGTCCCG-3';

[0117] Target of MSTN-gRNA2: 5'-TTTCCAGGCGAAGTTTACTG-3'.

[0118] Select two gRNA targets located on the FNDC5 gene:

[0119] Target of FNDC5-gRNA1: 5'-TGTACTCAGTGTCCTCCTCC-3';

[0120] Target of FNDC5-gRNA2: 5'-GCTCTTCAAGACGCCTCGCG-3'.

[0121] Primers used to amplify target-containing fragments are:

[0122] MSTN-F896: 5'-TCTCTCAGACAGTGCAGGCATTA-3';

[0123] MSTN-R1351: 5'-CGTTTCCGTCGTAGCGTGATAAT-3'.

[0124] FNDC5-F209: 5'-CAGTTCTCACTTGATGGCCTTGG-3';

[0125] FNDC5-R718: 5'-AGGGGTCTGGGGAGGAATGG-3'.

[0126] 1. Preparation of recombinant plasmids

[0127] 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.

[0128] MSTN-1S and MSTN-1A were synthesized separately, t...

Embodiment 3

[0154] Example 3, Screening of ADA Gene Knockout Targets

[0155] 1. Conservative analysis of preset targets of ADA gene knockout and adjacent genome sequences

[0156] Pig ADA gene information: encoding adenosine deaminase; located on chromosome 17;

[0157] GeneID is 100625920, Sus scrofa. The protein encoded by the porcine ADA gene is shown in SEQ ID NO:4. In the genomic DNA, the porcine ADA gene has 12 exons, wherein the 4th exon and its upstream and downstream sequences of 500 bp are shown in SEQ ID NO:5.

[0158] Using the genomic DNA of 8 pigs as a template, PCR amplification was performed using primer pairs consisting of primers ADA-GT-F259 / ADA-GT-R1005, followed by electrophoresis, see Figure 7 . The PCR amplification products were recovered and sequenced, and the sequencing results were compared with the ADA gene sequence in the public database. According to the comparison results, primers for detecting mutations were designed (the primers themselves avoided po...

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Abstract

The invention discloses a method for preparing recombinant cells derived from severe immunodeficiency pigs with combined knockout of four ADDI genes and a special kit thereof, in particular to a method for preparing four genes of ADA gene, DQA gene, DRA gene and IL2RG gene The method, sgRNA combination, plasmid combination and kit for combined knockout severe immunodeficiency pig-derived recombinant cells. The present invention also provides a sgRNA combination, consisting of sgRNA shown in SEQ ID NO: 11 ADA‑g7 , sgRNA shown in SEQ ID NO: 21 DQA‑gn2 , sgRNA shown in SEQ ID NO: 28 DRA‑g1 and the sgRNA shown in SEQ ID NO: 40 IL2RG‑g7 composition. The sgRNA combination can be used to: prepare recombinant cells; prepare immunodeficiency animal models. The invention lays a solid foundation for the preparation of severe immunodeficiency pig models, and has great application value for the research and development of severe immunodeficiency drugs.

Description

technical field [0001] The invention relates to a method for preparing recombinant cells derived from severe immunodeficiency pigs with joint knockout of four ADDI genes and a special kit thereof, in particular to a method for preparing four gene combinations of ADA gene, DQA gene, DRA gene and IL2RG gene. Methods, sgRNA combinations, plasmid combinations and kits for knockout severe immunodeficiency porcine-derived recombinant cells. 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,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/113C12N15/85C12N15/65A01K67/027
CPCC12N5/0656C12N15/1138C12N15/85C12N15/65A01K67/0273C12N2310/20C12N2510/00A01K2207/15A01K2217/075A01K2227/108A01K2267/0387
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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