Human huntingtin gene knock-in recombinant vector and its construction method and application in the construction of model pigs

Abstract

The invention discloses a recombinant vector for the knock-in of a human Huntington gene, a construction method of the recombinant vector and application of the recombinant vector in the constructionof a model pig. According to the recombinant vector, a human mutated Huntington exon gene is knocked in a fixed point manner for the first time, a virulence gene is knocked in by virtue of a CRISPR / Cas9 technique for the first time, and a donor vector is optimized by optimizing sfRNA, so that the probability that the gene is knocked into a positive cloned cell is increased; and by combining with apig cell nucleus transplantation technique, the probability that a directly obtained positive cell is knocked into the pig is increased, and a small human Huntington gene knock-in pig is obtained, thereby proving the efficient feasibility of the method for constructing gene modified pigs. The constructed Huntington gene knock-in model pig has behavioral characteristics such as respiratory disturbance and dyskinesia similar to human Huntington diseases, and stable heritable passage can be realized, so that a reliable model is provided for the research of the human Huntington diseases; and thenumber can be guaranteed so as to realize drug screening, gene treatment, stem cell treatment and the like, and the model can be a good human disease model.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant vector for knocking in human huntingtin gene, its construction method and its application in the construction of model pigs. Background technique [0002] Huntington's disease (Hutington Diseases, HD) is produced by N-terminal fragments carrying more than 37 polyglutamic acid repeats in the nucleus to form inclusion bodies and aggregates of nerve fibers (Gusella et al., Archives of neurology, 1993; Vonsattel et al., Journal of neuropathology and experimental neurology 1998), an autosomal dominant genetic disease caused by mutations in the Huntingtin gene (Htt) located on the short arm of chromosome 4, mainly manifested in the presence of mutated proteins in brain nerve cells accumulate and cause neuronal cell death. The cause of Huntington's disease is very clear. When the HTT gene contains more than 37 glutamine sequences, the mutant huntingtin protein will be m...

AI Technical Summary

Problems solved by technology

Although the use of Cas9 can produce high gene knockout efficiency, the efficiency of directly injecting Cas9, sgRNA, and recombinant vectors into fertilized eggs to produce gene knockout animals is far lower than that of gene knockout

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