动物造模 z-bio 2022-11-28 363

　　Objective: To establish a culture system for in vitro culture of adult marmoset kidney cells, and to initially apply the Cas9 editing system to the cultured marmoset kidney cells to establish a system for detecting sgRNA cleavage activity.

　　METHODS: Adult marmoset kidneys were harvested, and marmoset kidney cells were obtained by adhering and digestion methods. The cells were subjected to growth curve determination, transfection reagents (Viafect and Lipo2000) and resistance (puromycin and blasticidin). ) concentration screening, the SIRT1 sgRNA plasmid and Cas9 plasmid were co-transfected into marmoset cells, the genome was extracted and the gene-modified target fragment was amplified, and the amplified product was sequenced.

　　Results: The in vitro culture system of marmoset kidney cells was established; the transfection rate of ViaFect transfection reagent was more than 60%; the appropriate concentration of puromycin 4 μg/mL and blasticidin 8 μg/mL for cell resistance screening; A mutation peak began to appear near the PAM sequence of the sgRNA, which proved that the sgRNA successfully introduced the mutation.

　　Conclusion: The in vitro culture, cell transfection and resistance screening system of marmoset kidney cells were successfully established; CRISPR/Cas9 can be used for the editing of marmoset kidney cells, providing a basis for the selection of genetically modified marmoset targets.