Construction of POLQ Gene Knockout 293T Cell Line and Its Application for Target Integration of Exogenous Gene
Objective:To construct POLQ gene knockout lines in 293T cells using CRISPR/Cas9 technology and to investigate its application for targeted integration of foreign genes.Methods:By constructing pX330-sgRNA targeting vector targeting POLQ gene,then after T7 endonuclease Ⅰ verification,the vector with the highest targeting efficiency was selected to transfect 293T cells.Deletion of POLQ gene was confirmed by flow cytometric sorting,stable single cell clone culture,TA clone sequencing and Western blotting analysis.The GFP/mCherry homologous reporter vector targeting the AAVS1 and FBL sites was used to evaluate the efficiency of targeted integration of the exogenous gene in the knockout 293T POLQ-/-cell line,and the targeted integration of the donor reporter vector into the genome was verified by sequencing.Finally,the effects of POLQ gene knockout 293T cells on cell proliferation and survival rate were evaluated.Results:The POLQ gene was stably knocked out in 293T cells.Validation by reporter gene assays demonstrated that the efficiency of target integration of exogenous genes in the knockout cell line increased by an average of 2-fold to 7-fold.Furthermore,knocking out the POLQ gene had no significant effect on the proliferation and survival rate of 293T cells.Conclusion:Using CRISPR/Cas9 technology,a stable POLQ gene knockout cell line was successfully established in 293T cells,and the efficient targeted integration of exogenous genes was achieved.These findings provide a basis for using the cell line to generate stable integration of foreign genes,including antibody genes,facilitating the advancement of engineered cellular systems.
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