动物造模,基因敲除 z-bio 2022-07-04 256

　　Objective: To use CRISPR/Cas9 technology to knock out the Bmp9 gene fragment in the mouse genome to construct Bmp9 knockout mice. Methods According to the exon sequence of the Bmp9 gene, a sgRNA was designed and synthesized. The sgRNA was transcribed in vitro and mixed with Cas9 mRNA to display The fertilized egg cells were microinjected, and the injected fertilized egg cells were transplanted into the recipient animals to obtain offspring mice. The offspring mice were extracted for genomic DNA sequencing to identify their genotypes. The mice with correct genotypes were mated with the wild type to screen for homozygous small mice Mice. At the same time, the heart, liver, spleen, lung, and kidney of homozygous mice 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. Mutant mice were obtained after microinjection and replantation. After continuous mating, F2 homozygotes were obtained. The sequencing results showed that there were two genotypes in the mutant mice, one was 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: BMP9 knockout mice were successfully constructed using CRISPR/Cas9 technology.