The invention relates to the technical field of construction of conditional
gene knockout models, and discloses a construction method of a mouse Oct4 conditional
gene knockout model based on quadruple sgRNA collaborative targeting, which comprises the following steps: step 1, designing four sgRNAs, namely sgRNA1, sgRNA2, sgRNA3 and sgRNA4, in which the sgRNA1 and the sgRNA2 target 5'end and 3 'end non-coding regions of a second
exon of an Oct4
gene, and the sgRNA3 and the sgRNA4 target structural domain conserved sequences; step 2, preparing an electrotransfection solution, wherein the electrotransfection solution is composed of Cas9mRNA, the four sgRNAs and an electrotransfection
buffer solution; step 3, carrying out
electroporation treatment on the fertilized eggs of the mice; step 4, culturing the fertilized eggs subjected to the electrotransformation treatment to a
blastocyst stage; and 5, carrying out
genotype identification on the cultured
blastocyst. According to the scheme, the sgRNA combination and
embryo electrotransfection
system is optimized, the mouse Oct4 conditional
gene knockout model is efficiently and accurately constructed, a reliable tool is provided for regulation mechanism research of Oct4 in mouse early
embryo trigerm differentiation, and the medical transformation value of Oct4 is excavated.