动物造模,蛋白9基因敲除小鼠 z-bio 2021-06-25 238

　　Purpose: Do you want to use CRISR/Cas9 technology to knock out the Bmp9 gene fragment in the mouse genome and construct Bmp9 knockout mice?

　　The "method" is designed to synthesize sgRNA fragments based on the exon sequence of the Bmp9 gene. In vitro transcription, mixing sgRNA and Cas9 mRNA. After microinjecting fertilized egg cells, can the injected fertilized egg cells be transplanted into the recipient animal to obtain offspring? Is the genomic DNA of offspring mice extracted and sequenced to identify their genotypes? Do you want to screen homozygous mice for genotyping and wild-type mating? At the same time, the heart, liver, spleen, lung, and kidney of homozygous mice were collected and homogenized with total RNA and total protein to detect the expression of BMP9 in each tissue. qPCR, WB and immunohistochemistry?

　　Result: After 20bp sgRNA design and synthesis, in vitro transcription, microinjection, and re-transplantation to obtain genetically mutant mice, and continuous mating to obtain F2 homozygotes? , One is a 5 bp deletion mutation, and the other is a 13 bp deletion plus 1 bp insertion mutation? Do the qPCR, WB and immunohistochemical results show that the expression of BMP9 in the liver of knockout mice is significantly lower than that of wild-type C57BL/6?

　　Conclusion: Do you want to use CRISPR/Cas9 technology to successfully construct BMP9 knockout mice?