Generation of Stable α-ENaC Knockout Rat L2 Cell Strains by CRISPR/Cas9

doi:10.13560/j.cnki.biotech.bull.1985.2019-0919

Abstract

Abstract:

The purposes of this study are to generate α-ENaC knockout rat L2 cell line using CRISPR/Cas9 gene engineering technology and to explore the effect of α-ENaC on cell proliferation. CRISPR/Cas9 expression vectors with the α-ENaC gene knockout and the surrogate reporter were constructed on rat L2 cell line. After transfection and puromycin selection,the obtained mutational monoclones were confirmed by Western Blot and sequence analysis. CCK-8 method was used to determine the proliferation activity of the mutated cells. The CRISPR/Cas9 systems targeting the first exon of α-ENaC gene and the SSA-RPG surrogate reporter were designed and constructed successfully. Eight monoclones were picked after puromycin selection,and 2 of 8 monoclones showed significantly reduced α-ENaC expression and one of them lost the α-ENaC expression. The sequence analysis results confirmed that 2 of 3 monoclones were one single allelic mutation and the other one was double allelic mutation. In addition,there were no off-targets mutations. The proliferation activity of mutant cell lines decreased significantly,and that of the double allelic mutant cell line decreased more significantly. In sum,the α-ENaC knockout rat L2 cell line is successfully generated using CRISPR/Cas9 system combined with SSA-RPG surrogate reporters,and the α-ENaC gene is related to cell proliferation.

Key words: L2 cell, α-ENaC, CRISPR/Cas9, gene knockout

WANG Song, LI Peng-cheng, BAI Chao-hui, XU Hong-xin, YING Yan-min, ZHANG Mo, BAI Yi-chun. Generation of Stable α-ENaC Knockout Rat L2 Cell Strains by CRISPR/Cas9[J]. Biotechnology Bulletin, 2020, 36(3): 115-123.

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