Abstract:

Objective To knock out CXCR4 gene in 4T1 cell by CRISPR/Cas9 system and to construct 4T1 cell strain with knocking out CXCR4 gene stably. Methods Two pairs of sgRNAs that could specifically identify the exons of CXCR4 gene were designed to construct LentiCRISPRv2-sgRNA recombinant plasmid and transformed into competent Stbl3.Then the recombinant plasmids were screened for sequencing and transfected into 293T cell to packaging into lentivirus. After infection of 4T1 cells with lentivirus, stable transfected cells were selected by puromycin,and monoclonal cells were isolated and cultured by limiting dilution method . The genomic DNA of monoclonal cells was extracted and sequenced. The expression of CXCR4 mRNA was detected by Real-time PCR. The expression of CXCR4 protein was detected by Western blotting. Results The LentiCRISPRv2-sgRNA recombinant plasmid was successfully constructed, and a cell strain of knocking out CXCR4 deletion 27 bp was obtained. The expression level of CXCR4 mRNA was reduced, and there was almost no expression of CXCR4 protein in cell strain. Conclusion Recombinant plasmids targeting the CXCR4 gene was obtained by CRISPR/Cas9 gene edit system, and the cell strain with stable knockout CXCR4 gene was successfully screened out.

Key words: CRISPR/Cas9, Gene knockout, CXCR4, 4T1 cell, Real-time PCR, Western blotting