动物造模,基因敲除 z-bio 2021-11-12 764

　　Purpose: Use CRISR/Cas9 technology to knock out the Bmp9 gene fragment in the mouse genome to construct a Bmp9 gene knockout mouse. Method According to the exon sequence of the Bmp9 gene, design a segment of sgRNA and synthesize it. After sgRNA is transcribed in vitro and Cas9mRNA is mixed, it is displayed Microinjection of fertilized egg cells, transplant the injected fertilized egg cells to the recipient animal to obtain offspring mice. Extract the offspring mouse genomic DNA and sequence to identify its genotypes. The mice with the correct genotype identification are mated with wild-type mice, and then homozygous mice are selected. Rats. At the same time, the homozygous mouse heart, liver, spleen, lung, and kidney were taken, and the total RNA and total protein were extracted after homogenization, and the expression of BMP9 in each tissue was detected by qPCR, WB and immunohistochemistry.

　　Results: 20bp sgRNA was designed and synthesized and transcribed in vitro. After microinjection and replanting, genetic mutant mice were obtained. After continuous mating, F2 generation homozygotes were obtained. Sequencing results showed that the mutant mice had two genotypes, one It is a 5bp deletion mutation, and the other is a 13bp deletion accompanied by a 1bp insertion mutation. Compared with wild-type C57BL/6, the results of qPCR, WB and immunohistochemistry showed that the expression of BMP9 in the liver of knockout mice was significantly reduced.

　　Conclusion: The BMP9 knockout mouse was successfully constructed using CRISPR/Cas9 technology.