动物造模,基因敲除 z-bio 2021-11-12 359

　　Purpose: Use dual sgRNAs to construct miR-223 knockout mice.

　　Method: Design dual sgRNAs for miR-223 gene, and microinject the in vitro transcribed sgRNAs and Cas9mRNA into C57BL/6 mouse fertilized egg cells. After the mouse is born, its genomic DNA is PCR amplified and sequenced to identify the genotype. At the same time, the mouse liver was ground and the total RNA was extracted, and the expression of miR-223 in the liver was analyzed by real-time PCR.

　　Results: The miR-223 gene double sgRNAs were designed and transcribed in vitro. After purification, mice with fertilized egg cells were microinjected to obtain miR-223 gene mutant mice. Sequencing results showed that the mutant mice had 3 genotypes, one It is a 6bp deletion mutation, but has no effect on the miR-223 sequence; the other two are 162bp and 168bp deletion mutations, which completely delete the miR-223 precursor and mature region sequence. Compared with the wild type, these two kinds of mice The expression of miR-223 can hardly be detected in liver tissue.

　　Conclusion: Design dual sgRNAs and apply CRISPR/Cas9 technology to successfully construct miR-223 knockout mice.