动物造模,肌肉特异表达Cas9 z-bio 2021-07-01 549

　　Purpose: Obtain muscle-specific Cas9 protein mouse embryos through CRISPR/Cas9 technology, and lay the foundation for the establishment of a muscle-specific Cas9 mouse model.

　　Methods: Design mouse Rosa26 site sgRNA and verify its activity by in vitro enzyme digestion. At the same time, homologous recombination was used to construct muscle-specific homologous targeting vector; Rosa26sgRNA and Cas9 protein were injected into mouse embryos by microinjection, and PCR and sequencing were performed Detect the editing status of the embryo, and transplant it into the pseudo-pregnant female mouse at the same time, and wait for its production; The homologous targeting vector is injected into the mouse embryo together with Rosa26sgRNA and Cas9, and the integration is detected by PCR.

　　Results: In vitro enzyme digestion experiments showed that the combination of in vitro transcribed sgRNA and Cas9 protein can edit the target site; the muscle-specific homologous targeting vector Donor-SP-px459 was successfully constructed; Rosa26 gene-edited embryos were obtained by prokaryotic injection. Rosa26 gene-edited mice were obtained by transplantation; after injection of homologous targeting vectors, the muscle-specific gene targeting mouse embryos expressing Cas9 protein were successfully obtained.

　　Conclusion: Using CRISPR/Cas9 technology, we successfully obtained Rosa26 gene-edited embryos and mice, and obtained muscle-specific Cas9 protein-expressing mouse embryos, laying the foundation for the use of gene targeting to construct a muscle-specific Cas9-expressing mouse animal model.