Highly Efficient BLG Knockout in Bovine Mammary Epithelial Cells by Using CRISPR/Cas9
This study aimed to establish CRISPR/Cas9 mediated BLG-knockout(BLG-KO)sys-tem in bovine mammary epithelial cells(bMECs),and to explore the function effect of BLG-KO on bMECs.Three sgRNAs were designed from the first exon of the bovine BLG gene sequence and screened by using in vitro cleavage test.The Cas9 expression vector and sgRNAs vectors were cotransfected into bMECs by electroporation.Monoclonal cell lines were screened with 1.8 μg·mL-1 puromycin and 6 μg·mL-1 blasticidin.BLG-KO gene editing type were verified by using PCR amplification and sequencing analysis.The potential off-target sites were selected ac-cording to the sgRNA sequence and detected by PCR sequencing.The BLG expressing level of knockout cell was detected by Western blot(WB).Cell viability was tested by cell counting kit-8(CCK-8)assay,further the growth curve was drawn to analyze the proliferation effect of bMECs cells after BLG-KO.Two sgRNAs were screened and used for further gene edit.10 monoclonal cell lines were obtained after screening and 9 BLG-edit lines verified by PCR sequencing,4 clones with large fragment deleted at BLG sequence.Sequence analysis showed that the editing sites contained multiple gene-editing types,including insertion,deletion and base substitution.WB re-sult showed that BLG-KO cell lines with low BLG protein expressing level compare to none edit cell.Furthermore,the BLG-KO promote cell growth and proliferation according to the results of cell growth curve and CCK-8 assay.In this study,we constructed an efficient BLG-KO method in bMECs by using CRISPR/Cas9 system,and the results demonstrated that BLG-KO signifi-cantly affected the cell proliferation in bMECs,the results provided a cellular model for exploring the functional mechanism for BLG-KO cattle.
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