Severe immunodeficiency swine-derived recombinant cell, and preparation method and kit thereof

A technology for recombining cells and immunodeficiency, applied in biochemical equipment and methods, recombinant DNA technology, cells modified by introducing foreign genetic material, etc., can solve problems such as inability to conduct animal experiments, immune rejection, etc.

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

[0004] 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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  • Severe immunodeficiency swine-derived recombinant cell, and preparation method and kit thereof
  • Severe immunodeficiency swine-derived recombinant cell, and preparation method and kit thereof
  • Severe immunodeficiency swine-derived recombinant cell, and preparation method and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment 1, the preparation of plasmid

[0093] 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.

[0094] 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.

[0095] The plasmid pKG-U6gRNA was prepared, as shown in SEQ ID NO:3.

[0096] Plasmid pX330, plasmid pKG-GE3, and plasmid pKG-U6gRNA are all circular plasmids.

[0097] 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

[0101] Example 2, Screening of Targets for IL2RG Gene Knockout

[0102] 1. Conservation analysis of predetermined targets of IL2RG gene knockout and adjacent genome sequences

[0103] Pig IL2RG gene information: codes interleukin 2 receptor subunit gamma; located on X chromosome; GeneID is 397156, Sus scrofa. The protein encoded by the porcine IL2RG gene is shown in SEQ ID NO:16. In the genomic DNA, the porcine IL2RG gene has 9 exons, wherein the 4th exon and its upstream and downstream sequences of 500 bp are shown in SEQ ID NO: 17.

[0104] Using the genomic DNA of 8 pigs as templates, the primer pair consisting of primers IL2RG-GT-F4543 / IL2RG-GT-R5180 was used for PCR amplification, followed by electrophoresis, see Figure 5 . The PCR amplification products were recovered and sequenced, and the sequencing results were compared with the IL2RG gene sequence in the public database. According to the comparison results, primers for detecting mutations were designed (the prim...

Embodiment 3

[0185] Example 3, Screening of Targets for RAG1 Gene Knockout

[0186] 1. RAG1 gene knockout predetermined target and conservation analysis of adjacent genome sequences

[0187] Pig RAG1 gene information: encoding recombination-activating protein 1; located on chromosome 2; GeneID is 397506, Sus scrofa. The protein encoded by the porcine RAG1 gene is shown in SEQ ID NO:4. In the genome DNA, the porcine RAG1 gene has 2 exons, wherein the sequence of the second exon is shown in SEQ ID NO:5.

[0188] Using the genomic DNA of 8 pigs as a template, PCR amplification was carried out with a primer pair consisting of primers RAG1-GT-F4699 / RAG1-GT-R5306, followed by electrophoresis, see Figure 8 . The PCR amplification products were recovered and sequenced, and the sequencing results were compared with the RAG1 gene sequence in the public database. According to the comparison results, primers for detecting mutations were designed (the primers themselves avoided possible mutation s...

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Abstract

The invention discloses a severe immunodeficiency swine-derived recombinant cell and a preparation method thereof. Specifically, the invention relates to a severe immunodeficiency swine-derived recombinant cell with joint knockout of three genes such as an IL2RG gene, an RAG1 gene and an RAG2 gene, a method for preparing the recombinant cell, and a kit for preparing the recombinant cell. The invention provides an sgRNA (small guide ribonucleic acid) combination. The sgRNA combination is composed of sgRNAIL2RG-g7, sgRNARAG1-g4 and sgRNARAG2-g2. The target sequence binding region of the sgRNAIL2RG-g7 is shown as nucleotides at the first to the twentieth site in SEQ ID NO: 24. The target sequence binding region of the sgRNARAG1-g4 is shown as nucleotides at the first to the twentieth site inSEQ ID NO: 9. The target sequence binding region of the sgRNARAG2-g2 is shown as nucleotides at the first to the twentieth site in SEQ ID NO: 13. The invention lays a solid foundation for preparationof a severe immunodeficiency swine model, and has important application values for research and development of severe immunodeficiency medicine.

Description

technical field [0001] The invention relates to severe immunodeficiency pig-derived recombinant cells and a preparation method thereof. Specifically, the present invention relates to severe immunodeficiency porcine-derived recombinant cells in which the IL2RG gene, RAG1 gene and RAG2 gene are jointly knocked out, a method for preparing the recombinant cell, and a reagent for preparing the recombinant cell box. 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. Mo...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/113C12N5/10A01K67/027
CPCC12N15/8509C12N15/1138C12N15/113C07K14/7155C07K14/4705A01K67/0276C12N2310/20A01K2207/15A01K2217/075A01K2227/108A01K2267/0325
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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