Construction and Transfection Efficiency Evaluation of Goat CDK2 Gene Plasmid by CRISPR/Cas9 System
CDK2 is a necessary cell cycle regulatory kinase for cells to pass through and enter the S phase.It was reported that CDK2 gene is associated with the growth of many cancer cells.However,there were currently no reports on CDK2 gene knockout and related functions in goats.To address this issue,this present study aimed to construct and transfect the CDK2 plasmid by CRISPR/cas9 system.Firstly,the sgRNA fragment of the CDK2 gene was designed.After synthesizing the CDK2 sgRNA nucleotide sequence,a plasmid PYSY-CDK2 sgRNA was constructed that can simultaneously express sgRNA and Cas9 D10A.The plasmid was connected and transformed into E.coli DH5α cells to validate the transformants.Finally,it was transfected to HEK293T cells to detect cell fluorescence and proliferation.The results of enzyme digestion and sequencing confirmed that the construction of the CDK2 gene knockout vector was correct.Fluorescence microscopy detection showed that the transfection efficiency of cells was above 75%.Cell proliferation detection showed that the goat CDK2 knockout plasmid did not affect the proliferation of human cells.It was indicated that the vector constructed using CRISPR/Cas9 could provide a theoretical basis for obtaining goat CDK2 gene deficient cell lines and studying the molecular mechanism of cell apoptosis caused by Mycoplasma pneumonia infection.
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