关键词: CRISPR/Cas9系统, CACYBP/SIP, MCF-7细胞

Abstract: Objective To construct the CACYBP/SIP-/- MCF-7 cell strain and study the changes of its proliferation. Methods The specific sgRNA sequence was designed and Lenti-CAS9-sgRNA-puro plasmid was constructed, MCF-7 cells were transfected with the plasmid, and puromycin resistance was used to screened for positive clone. Cruiser was used to detect sgRNA activity, monoclonal strain was screened by limiting dilution method, and CACYBP/SIP protein expression was detected by Western blot, Brdu and MTT kits were used to detect cell proliferation after CACYBP/SIP gene deletion. Results The sequencing results showed that the Lenti-CAS9-sgRNA-puro plasmid was successfully constructed. The optimal screening concentration of puromycin was 0.9μg/mL. The results of Cruiser assay showed that sgRNA1 activity was the highest, and the obtained monoclonal strains was sequenced only. MTT and Brdu results showed that the proliferation of CACYBP/SIP-/- MCF-7 cell strain was weakened (P<0.05).Conclusion CACYBP/SIP-/- MCF-7 cell strain is successfully obtained and it’s proliferation ability is decreased.

Key words: CRISPR/Cas9 system, CACYBP, MCF-7