Construction Of CRISPR/Cas9 Lentiviral Vector Knockout MyD88 Gene Of Human Hepatocellular Carcinoma Cell Line

Objective: 1.SMMC-7721-Cas9 cells with stable expression of Cas9 protein and SMMC-7721-My D88-/-/My D88+/+pool cells were obtained by SMIS-7721 cells transfected with CRISPR / Cas9 lentiviral vector to build CRISPR / Cas9 gene editing platform.2.Screening and knockout identification of SMMC-7721-My D88-/-monoclonal cells.Methods: 1.Building CRISPR / Cas9 gene editing platform:(1)Designed and synthesized My D88 gene-specific 3 pairs of sg RNA(sg RNA-1,sg RNA-2,sg RNA-3)to connecte to the expression vector GV375.The recombinant plasmid and helper plasmid was co-transfected into 293 T intracellularly,resulting in lentiviral particles,and detecte the virus titer by fluorescence.(2)Determine the optimal MOI for lentivirus infection,and the optimal dose of puromycin screening.(3)SMMC-7721 cells were infected by Lenti-Cas9-puro lentivirus to obtain SMMC-7721-Cas9 cells through puromycin screening and SMMC-7721-Cas9 cells were infected with Lenti-sg RNA-Cherry virus to obtain SMMC-7721-My D88-/-/ My D88+/+ pool cells,of identificating by PCR,sequencing and Western blot at the gene and protein levels.2.Screening and knockout identification of SMMC-7721-My D88-/-monoclonal cells The monoclonal cells was screened by limited dilution method,extracting at the gene and protein level by PCR,sequencing,TA cloning and Western blot.Results: 1.Building CRISPR / Cas9 gene editing platform:(1)Sequencing confirmed that GV375 lentivirus recombinant expression vector was successfully constructed.(2)Recombinant expression vector and helper vector were co-transfected into 293 T cells,resulting in lentivirus particle titer of 1 × 108 TU / ml or more.(3)The efficiency of lentivirus infection was 92.27%,88.58% and 60.03% respectively when the MOI was 100,10,1,and the optimal MOI was 10;and the optimal screening dose of puromycin was 3μg / ml.(4)SMMC-7721-Cas9 cells and SMMC-7721-My D88-/-pool cell model were successfully constructed.Sequencing of SMMC-7721-My D88-/-pool cells appear peak and the expression of My D88 protein was significantly lower than that of normal and empty control.2.Screening and knockout identification of SMMC-7721-My D88-/-monoclonal cells Successful selection of SMMC-7721-My D88-/-monoclonal cells was performed by sequencing to show that a base A was inserted at the knockout target and the TA clone to shown that to be a homozygous clones of knocking out same base of allele two strands The expression of My D88 gene in SMMC-7721-My D88-/-cells was not expressed,but the expression of My D88 gene was not expressed in the normal and empty control groups at the protein level.Conclusions: 1.Successful building CRISPR / Cas9 lentiviral vector and transfected to SMMC-7721 cells to obtain SMMC-7721-Cas9 cells with stable expression of Cas9 protein and SMMC-7721-My D88-/-/My D88+/+pool cells,which provide experimental basist to knockout other gene of SMMC-7721 cells.2.Successful selection of knockout My D88 allele of SMMC-7721-My D88-/-homozygous monoclonal cells,which provided experimental basis for studying the mechanism of My D88 gene on human hepatocellular carcinoma cell line.