关键词: CRISPR/Cas9, 溶酶体运输调节因子(Lyst)基因, 基因敲除

Abstract: Objective To obtain the animal model of NK cell deficient mice and study the function of the lysosomal trafficking regulator (Lyst) gene.Method The CRISPR/Cas9 technique was used to knockout Lyst gene on C57BL/6 mice.On fiftieth exons of mouse Lyst gene coding region,CRISPR/ Cas9 target primer (sgRNA) was designed pUC57-Lyst-sgRNA vector was constructed.The recombinant plasmids and Cas9 plasmids were transcribed into mRNA in vitro separately by using T7 RNA polymerase.mRNA was microinjected to mouse fertilized egg proportionally.We use embryo transplant to obtain Lyst knockout mice.PCR amplification and sequencing were used to screen the individual animal of Lyst gene change.Result Seven F0 generation knockout mice were obtained after embryo transplant with the coat color change obviously.Conclusion Lyst gene-deficient mice can be constructed by using CRISPR/Cas9 technology.The phenotype in morphology is consistent with those reported in the literature.

Key words: CRISPR/Cas9, Lysosomal trafficking regulator (Lyst) gene, Gene knockout