Establishment of a stable Beclin1 gene knockout ST cell line using CRISPR/Cas9 technology
Yu Hui
Chen Haien
Su Qiling
Huang Yidan
Wu Jiang
Kang Kai
Abstract：This study aimed to utilize CRISPR/Cas9 gene editing technology to knockout the Beclin1 gene in swine testis(ST)cell lines,thereby establishing an autophagy gene knockout cell line.This provides a foundation for subsequent research on the relationship between autophagy and other physiological functions during ST cell proliferation.According to CRISPR/Cas9 target design principles,specific sgRNAs targeting sequences in the first exon of the Beclin1 gene were designed,and the pLV3-U6-MCS-sgRNA recombinant plasmid was constructed.After sequencing verification,the recombinant plasmid was transfected into ST cells.Puromycin selection and limiting dilution were employed to obtain monoclonal cell lines,with Beclin1 knockout efficiency confirmed by Western blot analysis.The results demonstrated that stable ST cell lines with Beclin1 knockout were successfully obtained following CRISPR/Cas9 system treatment.Compared with the original ST cell line,no detectable expression of Beclin1 protein was observed in the stable knockout cell line of Group B.This study confirms the successful establishment of Beclin1-knockout ST cell lines,providing a valuable tool for functional research on Beclin1.
Keywords：CRISPR/Cas9Beclin1ST cellsGene knockout
Publication Date：2025-06-15
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：5( 18-22 )