Establishment Of An IGF-ⅡR Gene Site-integrated Skbr3 Cell Line Using CRISPR/Cas9

Objective The purpose was to establish SKBR3 cell line with IGR-IIR gene site-integrated using CRISPR/Cas9 system,and to provide cell model for exploring the effect of IGF-IIR on trastuzumab resistance in HER-2 positive breast cancer cells.Methods Six pairs of sg RNA were designed and synthesized according to AAVS1 gene sequence.UCA^TMCRISPR/Cas9 rapid construction and activity detection kit were respectively connected with p CS vector to screen the most efficient sg RNA to construct Cas9/sg RNA expression vector.IIGF-IIR fragment was ligated into h AAVS1-KI vector with Asi SI+Bstz17I digestion,and IGF-IIR targeting vector was constructed.Eco RI+Sca I,Nco I,Bgl II digestion identification and sequencing verification;flow cytometry was used to determine the best electrotransfection For optimal conditions,Cas9/sg RNA vector and IGF-IIR targeting vector were co-transfected into SKBR3 cells under optimal electrotransformation conditions;SKBR3 was treated with puromycin at different concentrations,and all cells were killed in 7 days as a standard.After determining the optimal screening concentration Screening,extracting the genome of the mixed cells,performing PCR identification and sequencing detection,and constructing a mixed clone cell line for site-integrated integration of the IGF-IIR gene;monoclonal preparation using semi-solid medium method and limiting dilution method.Results Cas9/sg RNA vector acting on the target sequence of the AAVS1 gene was successfully constructed.sg RNA activity test showed that sg RNA2 had the highest efficiency.IGF-IIR fragment digested by Asi SI and Bstz17I was ligated into h AAVS1-KI vector,and Eco RI and Scal were used for dual enzyme identification and sequencing to confirm the successful construction of IGF-IIR targeting vector;the optimal conditions for electrotransfection were 1200v,20ms,and 2 pulses;the optimal screening concentration of puromycin was 0.5μg/m L;IGF-IIR targeting vector and Cas9/sg RNA2 vector were successfully transfected into HER-2 positive breast cancer cells SKBR3.PCR identification and sequencing confirmed that the genotypes of mixed cloned fragments were correct;the growth of monoclonal SKBR3 were inhibited due to poor cell status,gradual apoptosis,and IGF-IIR high expression in vitro.Conclusion The mixed cloned cells with IGF-IIR gene integrated at the AAVS1 site was successfully obtained,which layed the foundation for further exploring the effect of IGF-IIR on trastuzumab resistance in HER-2 positive breast cancer cells.