SIRT2稳定敲除细胞系的建立及对组蛋白修饰的影响

Generation of SIRT2 Stably Knockout Cell Lines and its Effect on Histone Modification

目的利用CRISPR/Cas9系统构建稳定敲除SIRT2基因的HEK293细胞系,研究其对组蛋白不同位点的修饰情况。方法构建lentiCRISPR v2-sgRNA SIRT2敲除质粒,通过慢病毒包装,感染HEK293细胞,嘌呤霉素筛选出阳性克隆。对筛选的细胞株进行T7E1验证,来验证所设计的sgRNA的有效性。应用蛋白质免疫印迹检测SIRT2蛋白水平,筛选得到SIRT2稳定敲除细胞系。使用蛋白质免疫印迹的方法在蛋白水平验证SIRT2对组蛋白乙酰化、甲基化的影响。结果通过测序证明lentiCRISPR v2-sgRNA SIRT2敲除质粒正确,T7E1验证了所构建的质粒具有良好的CRISPR/Cas9活性,使基因组碱基发生突变。蛋白质免疫印迹证明SIRT2敲除细胞系中SIRT2表达显著降低。在CRISPR/Cas9系统敲除SIRT2的HEK293细胞中,组蛋白H4乙酰化在赖氨酸第16位(H4K16ac)表达显著升高,组蛋白H4乙酰化在赖氨酸第5位(H4K5ac)表达下降,组蛋白H3二甲基化在赖氨酸第79位(H3K79me2)表达升高。结论获得SIRT2稳定敲除细胞系,便于后续SIRT2对组蛋白修饰功能的研究。

Objective SIRT2 knockout in HEK293 cell line was constructed with CRISPR/Cas9 to study histone modification at different sites.Methods The lentiCRISPR v2-sgRNA SIRT2 knockout plasmid was constructed in HEK293 cells by lentiviral packaging.Puromycin was used to purify the positive clones.The effectiveness of the designed sgRNA was validated by T7E1 in HEK293 cell lines.SIRT2 protein levels were detected by Western blot and the results confirmed that SIRT2 knockout cell lines were successful.Histone acetylation and methylation were testified in SIRT2+/+and SIRT2-/-HEK293cells by Western blot.Results SIRT2 knockout HEK293 cell lines were generated.T7E1 endonuclease cutting proved that the constructed plasmids possess ideal CRISPR/Cas9 activities and could lead to the basic mutation in genomic DNA.Western blot confirmed that SIRT2 expression was significantly decreased in SIRT2 knockout cell line.Histone 4 acetylation at lysine16(H4K16ac)was significantly elevated,protein levels of histone4 acetylation at lysine5(H4K5ac)was decreased and histone 3 dimethylation at lysine 79(H3K79me2)was elevated in SIRT2 knockout cell line.Conclusion Successful construction of SIRT2 knockout cell line facilitated the study of histone modification with SIRT2.