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
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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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