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Method for constructing severe immunodeficiency and liver injury dual pig model and application

A technology for recombinant plasmids and porcine fibrosis, applied in chemical instruments and methods, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of highly simulating physiological metabolic processes, low success rate of modeling, and inflammatory response Different problems, achieve huge biomedical research application prospects and market value, overcome efficiency problems, and reduce inflammatory reactions

Pending Publication Date: 2022-02-08
YUNNAN AGRICULTURAL UNIVERSITY
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  • Claims
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AI Technical Summary

Problems solved by technology

[0004] (1) Rodents such as mice are distantly related to humans, have large body size differences, and have short lifespans. It is difficult to simulate human physiological and metabolic processes with rodents such as mice. The application in the field of scientific research has been greatly restricted
[0005] (2) At present, the construction of dual mouse models uses more traditional gene editing methods, and even chemical drugs are used in the preparation of liver injury, which has irreversible damage, and the success rate of modeling is low, which is not conducive to subsequent simulations Human Surgical Procedures and Preclinical Evaluation
[0006] (3) FAH obtained by hybridization methods commonly used - / - / Rag - / - / IL2RG - / - A mouse model of simultaneous three-gene modification, first by Rag - / - / IL2RG - / - based on the FAH - / - Mice mated to produce Rag - / + / IL2RG - / + / FAH - / + individuals, and then obtain FAH by selfing - / - / Rag - / - / IL2RG - / - Individuals modified by the three genes at the same time, and some are even chimeric mice obtained by injection of fertilized eggs. This method takes a long time to construct and the production efficiency of the model is low.
[0007] (4) Current mouse FAH - / - / Rag - / - / IL2RG - / - In the model, there is no way to remove NK cells, and the inflammatory response is different from that of humans, so it cannot reshape the human model very well.

Method used

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  • Method for constructing severe immunodeficiency and liver injury dual pig model and application
  • Method for constructing severe immunodeficiency and liver injury dual pig model and application
  • Method for constructing severe immunodeficiency and liver injury dual pig model and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Pig RAG2 - / - / IL2RG - / Y / FAH - / - Construction of three gene targeting vectors

[0051] The nucleotide sequences of RAG2 (Gene ID: 100151744), IL2RG (Gene ID: 397156) and FAH (Gene ID: 100623036) genes of pigs were searched in the NCBI database. Exon 5 sequence, the second exon sequence of FAH gene, using online software (http: / / crispor.tefor.net / ) to design and screen the target gRNA site and connect the sgRNA to the backbone vector to obtain RAG2-gRNA (SEQ ID NO:1) targeting vector ( figure 2 ), further screened to obtain the optimal porcine IL2RG / FAH gene recombination targeting vectors, respectively IL2RG-gRNA (SEQ ID NO: 2), FAH-gRNA (SEQ ID NO: 3) ( image 3 ). The above targeting vectors were co-transfected into pig fetal fibroblasts and screened to obtain RAG2 - / - / IL2RG - / Y / FAH - / - Three-gene mutant porcine fetal fibroblast cell line used as donor cells for somatic cell nuclear transfer to construct RAG2 - / - / IL2RG - / Y / FAH - / - Three gene...

Embodiment 2

[0052] Example 2: RAG2 - / - / IL2RG - / Y / FAH - / - Construction of three gene-edited cloned pigs

[0053] 1. Design and screen targeting for the coding region of the porcine RAG2 (Gene ID: 100151744) gene, the fifth exon of the IL2RG (Gene ID: 397156) gene, and the second exon of the FAH (Gene ID: 100623036) gene The gRNA sites are RAG2-gRNA (SEQ ID NO:1), IL2RG-gRNA (SEQ ID NO:2), and FAH-gRNA (SEQ ID NO:3).

[0054] 2. Connect the gRNA sequence SEQ ID NO:1 of the RAG2 gene to the GL3-U6-sgRNA backbone carrier, and the recombinant plasmid RAG2-GL3-U6-sgRNA and pST1374-NLS-flag-linker-Cas9 with the correct sequence after sequencing verification The plasmids were co-transfected into porcine fetal fibroblasts (pST1374-NLS-flag-linker-Cas9:RAG2-GL3-U6-sgRNA=2:1) ​​by a nuclear transfer instrument, and a total of 9 single-cell clones were obtained after screening, of which 9 No. single-cell clone was RAG2 biallelic knockout, and RAG2KO-09 single-cell clone was further used as dono...

Embodiment 3

[0057] Example 3: RAG2 - / - / IL2RG - / Y / FAH - / - Genotype identification and phenotype analysis of three gene-edited cloned pigs

[0058] Genotyping. The genomic DNA of two surviving cloned piglets (RGFP19, RGFP20) was extracted, and the RAG2 / IL2RG / FAH gene mutations of the cloned piglets were identified by PCR, T7ENI, Sanger sequencing and other methods, and the results showed that they were all RAG2 - / - / IL2RG - / Y / FAH - / - Triple gene knockout ( Image 6 ), and further detected RAG2 - / - / IL2RG - / Y / FAH - / - No off-target was found in the three gene-edited cloned pigs (Table 1).

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Abstract

The invention relates to a method for constructing a severe immunodeficiency and liver injury dual pig model and application, and belongs to the technical field of animal biology. According to the method, RAG2, IL2RG and FAH genes in porcine fetal fibroblasts are knocked out by adopting a CRISPR / Cas9 technology, RAG2- / - / IL2RG- / Y / FAH- / -three-gene editing cloned pigs are constructed by utilizing a somatic cell nuclear transplantation technology, and the severe immunodeficiency and liver injury dual pig model is obtained through phenotype analysis and identification. The problems of long production cycle, low efficiency, irreversible injury, lack of immunodeficiency and non-ideal humanization degree of simultaneous application of liver injury and the like in the existing model construction technology are overcome, and batch construction of the severe immunodeficiency and liver injury dual pig model can be realized through a continuous cloning technology, and the method has huge advantages and potential market application prospects in the related fields of tumor biology, cell transplantation, humanized animal models and the like.

Description

technical field [0001] The invention belongs to the field of animal biotechnology, and in particular relates to a method and application for constructing a double pig model of severe immunodeficiency and liver injury. Background technique [0002] Immunodeficient animals play an important role in the fields of biomedical and basic medical research. Liver transplantation is an important treatment for end-stage liver failure, and the shortage of liver donors is the main bottleneck restricting the clinical application of liver transplantation. As one of the important ways to solve the serious shortage of liver organs, hepatocyte transplantation technology can produce humanized liver by implanting human hepatocytes in animal models of immunodeficiency and liver injury, and obtaining humanized hepatocytes in batches can solve the problem. The limited source of primary hepatocytes has played an important role in the treatment of human hepatitis B (HBV) and hepatitis C (HCV) disea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/12C12N5/077A01K67/027
CPCC12N15/8509C07K14/47C12N5/0656A01K67/0276A01K2227/108A01K2267/0387A01K2267/03C12N2310/20
Inventor 魏红江陈清烽赵恒角德灵赵红业
Owner YUNNAN AGRICULTURAL UNIVERSITY
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