Establishment and Application of Immortalized Cell Line of Mouse Embryonic Fibroblasts Derived from LSL-Cas9 Mice
Objective:To facilitate the detection of sgRNA splicing efficiency,lentiviral transfection of embryonic fibroblasts(MEF)from LoxP-Stop-LoxP(LSL)-controlled Cas9 transgene-carrying(LSL-Cas9)mice with simian virus 40(SV40)large T(LT)antigen was utilized to establish an immortalized LSL-Cas9 mouse MEF line.By transforming the plasmid carrying the sgRNA expression cassette tandem with the constitutive promoter-driven Cre expression cassette into the cell line,regulatory expression of Cas9 can be realized.This system overcomes the problem of low transfection efficiency caused by the large Cas9 sequence,and provides an economical,convenient and efficient tool to verify the sgRNA shearing efficiency.Methods:LSL-Cas9 fetal mice were used as experimental animals,and their MEF cells were isolated and infected with SV40 LT lentivirus;hygromycin was used to screen successfully transformed cells.Detection of fibroblast marker expression and cellular senescence by immunofluorescence and the integration of SV40 LT antigen genes in MEF was identified by polymerase chain reaction(PCR),and a cell line with unlimited proliferation of immortalization was established.The IL10Rα was used as a representative gene to verify the feasibility of this system.Four sgRNAs were designed and the immortalized MEF cell line from LSL-Cas9 mice was transformed using a plasmid containing a PU6-sgRNA wtih a tandem PCSP-Cre gene expression cassette.The sgRNA splicing efficiency was evaluated by the T7 Endonuclease I(T7EI)method,and the sgRNA with optimal targeting was selected.Results:Positive cells screened with hygromycin were expanded and stably passed to 60 generations,exhibiting the characteristics of immortalized,infinitely proliferating cell lines.The result of PCR identification showed that the SV40 LT antigen gene was stably integrated into the MEF genome.Conclusions:The SV40 LT antigen gene-mediated immortalized infinitely proliferating LSL-Cas9 MEF cell line was successfully established for efficient and rapid validation of sgRNA-guided Cas9 shear efficiency.
万方数据
维普
