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System for preparing severe immunodeficiency swine-derived recombinant cells with joint knockout of four RRIP genes

A technology of recombinant cells and immunodeficiency, which is applied in the system field of pig-derived recombinant cells for severe immunodeficiency, and can solve problems such as inability to conduct animal experiments and immune rejection

Active 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

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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  • System for preparing severe immunodeficiency swine-derived recombinant cells with joint knockout of four RRIP genes
  • System for preparing severe immunodeficiency swine-derived recombinant cells with joint knockout of four RRIP genes
  • System for preparing severe immunodeficiency swine-derived recombinant cells with joint knockout of four RRIP genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1, Preparation of Plasmids

[0103] Preparation of plasmid PX330-U6-chiMERIC_BB-CBH-HSPCAS9, as shown in SEQ ID NO: 1. Plasmid PX330-U6-chiMERIC_BB-CBH-HSPCAS9, referred to as plasmid PX330.

[0104] Preparation of plasmid pu6 géf1a-mnls-hspcas9-EGFP-PURO, as shown in SEQ ID NO: 2. Plasmid pu6 géf1a-mnls-HSPCas9-EGFP-PURO, referred to as plasmid pkg-ge3.

[0105] Preparation of plasmid PKG-U6GRNA, as shown in SEQ ID NO: 3.

[0106] Plasmid PX330, plasmid PKG-GE3, plasmid PKG-U6GRNA are both annular plasmids.

[0107] Structure of plasmid PX330 figure 1 . SEQ ID NO: 1, 440-725 Nucleotide Composition CMV Reinforcement, Section 727-1208 Nucleotide Composition Chickenβ-Actin promoter, No. 1304-1324 Nucleotide encoded SV40 nuclear positioning signal (NLS ), No. 1325-5449 Nucleotide encoding Cas9 protein, race 5450-5497 Nucleotide coded Nucleoplasmin nuclear positioning signal (NLS).

[0108] Structure of plasmid pkg-ge3 figure 2 . SEQ ID NO: 2, No. 395-680 Nucleotide C...

Embodiment 2

[0111] Example 2, comparison of plasmid PX330 and plasmid pkg-ge3

[0112] Select two GRNA targets located at the MSTN gene:

[0113] MSTN-GRNA1 target: 5'-gctgattgtgctggtcccg-3 ';

[0114] MSTN-GRNA2 target: 5'-TTTCCAGGCGAAGTTTACTG-3 '.

[0115] Select two GRNA targets located in the FNDC5 gene:

[0116] Targets of FNDC5-GRNA1: 5'-TGTACTCAGTGTCCTCCTCCC-3 ';

[0117]Targets of fndc5-gRNA2: 5'-gctcttcaagacgcctcgcg-3 '.

[0118] The primers used to amplify fragments containing targets are:

[0119] MSTN-F896: 5'-TctCAGACAGTGCAGGCATTA-3 ';

[0120] MSTN-R1351: 5'-cgtttccgtcgtagcgtgataat-3 '.

[0121] FNDC5-F209: 5'-cagttctcacttgatggcctTGG-3 ';

[0122] FNDC5-R718: 5'-aggggtggggaggaatgg-3 '.

[0123] First, prepare recombinant plasmids

[0124] Substrate PKG-U6GRNA was taken to use restriction endonuclease BBSI to recover the carrier skeleton (about 3 kb linear block).

[0125] MSTN-1s and MSTN-1A were synthesized, and then mixed and annealing was mixed to give a double-stranded DNA m...

Embodiment 3

[0151] Example 3, Screening of targets of Rag1 gene knockout

[0152] I. Rag1 gene knockout preset target and neighbor genomic sequence conservative analysis

[0153] Pig RAG1 gene information: Code Recombination-Activating Protein 1; Located in chromosome 2; GeneId is 397506, SUS Scrofa. Proteins encoded by pig RAG1 genes are shown in SEQ ID NO: 4. In the genomic DNA, the pig RAG1 gene has two exons in which the second exon sequence is shown in SEQ ID NO: 5.

[0154] The primer of the primer RAG1-GT-F4699 / RAG1-GT-R5306 is used in the primer of the primer RAG1-GT-F4699 / RAG1-GT-R5306, respectively, and then electrophoresis. Figure 7 . The PCR amplification product was recovered and sequenced, and the sequencing results were compared to the RAG1 gene sequence in the public database. Depending on the comparison result, primers (primer itself avoids possible mutation sites) are designed. The primer for detecting mutations is: RAG1-NF126 / RAG1-NR525.

[0155] RAG1-GT-F4699: 5'-ca...

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Abstract

The invention discloses a system for preparing severe immunodeficiency swine-derived recombinant cells with joint knockout of four RRIP genes, and particularly relates to a system for preparing severeimmunodeficiency swine-derived recombinant cells with joint knockout of four genes such as an RAG1 gene, an RAG2 gene, an IL2RG gene and a PRKDC gene. The invention provides an sgRNA (Ribonucleic Acid) combination. The sgRNA combination consists of sgRNARAG1-g4, sgRNARAG2-g2, sgRNAIL2RG-g7 and sgRNAAPRKDC-g6. The target sequence binding region of the sgRNARAG1-g4 is shown as nucleotides at the first to the twentieth site in SEQ 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 target sequence binding region of the sgRNAIL2RG-g7 is shown as nucleotides at the first to the twentieth site in SEQ ID NO: 24; and the target sequence binding region of the sgRNAPRKDC-g6 is shown as nucleotides at the first to the twentieth site in the SEQ ID NO: 34. 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 present invention relates to a system for preparing a severe immunodeficient pig source recombinant cells for the preparation of RRIP combined with knockout, specifically, for the preparation of RAG1 gene, RAG2 gene, IL2RG gene, and PRKDC gene joint knocking Semi-immunodeficient pig source recombinant cells system. Background technique [0002] Severe Combined Immunodeficiency, SCID is the most serious phenotype in primary immunodeficiency disease, refers to development, differentiation of T cells, B cells and NK cells caused by factors such as genetic, developmental or infection. , Proliferation, metabolism or dysfunction. In 1950, Glanzmann and Riniker reported the SCID disease in human infants. At the global, SCID's neonatal incidence is approximately 1 / 50000, and the age is early, the clinical manifestation is heavy, and the mortality is high. Most SCIDs are due to an abnormality of immune-related genes, while the main genetic approach of SCID inclu...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N15/62C12N15/12C12N15/54C12N5/10A01K67/027
CPCC12N15/1138C12N15/1137C12N15/113C12N15/8509C12N9/22C07K14/7155C12N9/12C07K14/4705C12N5/0656A01K67/0276C12Y207/11C12N2310/20C12Y207/11001C12N2800/107C12N2830/48C12N2830/50C07K2319/00C07K2319/09C07K2319/60C12N2510/00A01K2217/075A01K2227/108A01K2267/0387
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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