Recombination activating gene 2 gene targeting vector, production of SCID-like miniature pigs by TALEN-mediated gene targeting and use thereof
AssigneesKonkuk University Industrial Cooperation Corp., The Curators of the University of Missouri
InventorsJin-Hoi Kim, Deug-Nam Kwon, Randall S. Prather, Kiho Lee
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Abstract
There are provided to a Rag-2 (Recombination activating gene 2) gene targeting vector, a method for producing SCID-like miniature pigs introduced with the vector, and a use thereof.
Claims10
A method for producing severe combined immunodeficiency (SCID)-like miniature pigs having a recombination activating gene (RAG) 2-allelic mutation, the method comprising:
- inducing an allelic mutation in a pig RAG 2 gene by exposing the gene to a transcription activator-like effector nuclease (TALEN) that recognizes a TALEN-recognizing sequence region as set forth in SEQ ID NO. 1 in Chromosome 2 of the pig;
- producing mutant embryos by using cells comprising the induced allelic mutation, wherein the producing is performed by somatic cell nuclear transplantation (SCNT);
- and
- transferring the embryos to a surrogate gilt and allowing the embryos to gestate to term.
A method for sorting cells having an allelic mutation in a recombination activating gene (RAG) 2 gene, the method comprising:
- introducing to the cells a vector encoding a TAL effector-DNA nuclease capable of inducing one or more mutations in a TALEN-recognizing sequence region as set forth in SEQ ID NO. 1 in Chromosome 2 of a pig or surrounding sites thereof, and a reporter vector encoding a gene encoding a red fluorescent protein (RFP), a gene encoding a green fluorescent protein (GFP), a targeting sequence as set forth in SEQ ID NO. 1, and a H-2KK gene, and
- sorting the cells based on presence of the reporter vector.
A cell comprising an induced RAG 2 allelic mutation, wherein the RAG 2 allelic mutation is monoallelic or biallelic, and wherein the RAG 2 allelic mutation is RAG2+/Δ140, S141H or RAG2+/Δ140, S141H/Δ140-527.
Dependencies are inferred from the claim text.