动物造模,基因敲除 z-bio 2021-05-20 642

　　Purpose: Use CRISPR/Cas9 technology to construct miRNA-29b1 knockout mice.

　　Method: Design sgRNA, sgRNA and Cas9 for miRNA-29b1 gene, and microinject them into C57BL/6 mouse fertilized egg cells after in vitro transcription. After the mouse is born, the genomic DNA is sequenced to identify the genotype. At the same time, the mouse heart, liver, spleen, lung and kidney are collected. After crushing, the total RNA is extracted and the miRNA is analyzed by real-time PCR expression in these organs. -29b1.

　　Result: A 20bp miRNA-29b1sgRNA was designed and transcribed in Cas9 in vitro, and miRNA-29b1 mutant mice were obtained after microinjection of fertilized mouse eggs. The sequencing results showed that the mutant mice had two genotypes. One is a 10 bp deletion mutation, the other is a 22 bp deletion mutation, and the insertion mutation is 3 bp. Compared with wild-type mice, the expression of miRNA-29b1 in the heart, liver, spleen, lung, kidney and other tissues of genetically mutant mice was significantly reduced.

　　Conclusion: The miRNA-29b1 gene knockout mice were successfully constructed using CRISPR/Cas9 technology.