利用CRISPR/Cas9技术建立小鼠Bdh2、Dnmt3a和Dusp9敲除新型胚胎干细胞系

The Establishment of Bdh2、Dnmt3a and Dusp9 Knockout Advanced Embryonic Stem Cell Lines by CRISPR/Cas9 Technique

胚胎干细胞因其具有体外自我更新以及多向分化的能力,成为研究基因功能、细胞治疗和组织再生等的有力工具.胚胎干细胞的基因敲除模型在基因功能研究中起着重要作用.我们利用实验室现有的G3-AH和tWG3II-2两种胚胎干细胞系,通过脂质体转染嘌呤霉素(puromycin)标记的PX459-sgRNA载体,使用CRISPR-Cas9技术分别构建Bdh2、Dnmt3a和Dusp9敲除的胚胎干细胞系,通过基因型鉴定、PCR产物测序、qPCR和Western Blot等实验得到Bdh2、Dnmt3a和Dusp9纯合缺失的胚胎干细胞系.之后,qPCR、免疫荧光染色分别检测了Bdh2、Dnmt3a和Dusp9缺失的胚胎干细胞中多能基因、DNA甲基化相关基因和胚层分化相关基因表达变化.另外,通过嵌合体制备方法检测Dusp9基因敲除对胎儿卵黄囊(yolk sac)和胎盘(placenta)等发育的影响.实验结果表明,KO细胞系中分别敲除的基因(Bdh2、Dnmt3a和Dusp9)在mRNA水平和蛋白水平的表达均显著降低(p<0.05),且不同程度地影响了DNA甲基化相关基因和胚层分化相关基因的表达.Dusp9KO嵌合体结果显示,Dusp9KO均能贡献到胎儿及卵黄囊,但是,能否贡献到胎盘尚待进一步深入探究.本研究成功获得纯合敲除Bdh2、Dnmt3a和Dusp9基因的胚胎干细胞系,为研究Bdh2、Dnmt3a和Dusp9三种基因在胚胎干细胞中的作用提供了重要依据.

Embryonic stem cells(ESCs)is an important tool for gene function research,cell therapy and tissue regeneration research,due to their self-renewal and multidirectional differentiation ability.ESCs gene-knockout model plays important roles in the gene function study.In this study,embryonic stem cell lines(G3-AH and tWG3 II-2)were used to generate Bdh2,Dnmt3 aand Dusp9-knockout advanced stem cells(ASCs)and ESCs by liposome transfecting puro-resistant PX459-sgRNA expression plasmids with CRISPR/Cas9 system.The Bdh2,Dnmt3 aand Dusp9 homozygous deletion was confirmed by genotyping,PCR products sequencing,qPCR,and Western Blot,respectively.Then,qPCR and immunofluorescence staining were conducted to detect the relative expression changes of pluripotency genes,DNA methylation-related genes,and germ layers marker in Bdh2,Dnmt3 aand Dusp9-knockout ESCs.In addition,chimeras were prepared to examine the effects of Dusp9 gene knockout on the development of fetal,yolk sac and placenta.The results showed that,the expression of Bdh2,Dnmt3 aand Dusp9 in their knockout cell lines were significantly reduced(p<0.05),which influenced the expression of genes related to DNA methylation and germ layers marker in different levels.The results of Dusp9-knockout chimeras indicated that Dusp9-knockout ASCs could contribute to both fetal and yolk sac.However,whether they could contribute to placenta remained to be explore.In conclusion,Bdh2,Dnmt3 aand Dusp9 knockout embryonic stem cell lines were successfully established,which provided basic data for the study of Bdh2,Dnmt3 aand Dusp9 in embryonic stem cells.