miRNA-205过表达载体的构建及其对猪诱导性多能干细胞建系的作用

Construction of miRNA-205 overexpression vector and its effect on porcine induced pluripotent stem cell establishment

目的:构建miRNA-205过表达载体并研究miRNA-205对猪诱导多能干细胞系建立的影响。方法:对本实验室已建系的猪primed胚胎干细胞、猪内细胞团细胞及猪胎儿成纤维细胞三者之间进行miRNA表达谱的分析比较,利用PCR扩增miRNA-205前体序列,并将其克隆于基础表达载体p CAGDNA3-h Fat1,构建过表达载体p CAG-miR205。使用脂质体转染技术将该载体转染到已转基因修饰的猪胎儿成纤维细胞(含有鼠源干细胞多能性转录因子Oct4、Sox2、Klf4和c-Myc)。利用干细胞培养液诱导培养建立猪诱导性多能干细胞系。通过实时荧光定量PCR检测转染前后细胞内miRNA-205的表达情况,比较分析miRNA-205对猪诱导性多能干细胞的建系效率的影响。结果:成功构建了p CAG-miR205过表达载体;相较于未转染细胞,转染该载体细胞的miRNA-205表达量显著升高(P<0.01);利用干细胞培养液诱导后培养转染组的诱导性多能干细胞克隆长出率高于未转染细胞组。结论:研究结果表明在细胞水平上过表达micro RNA-205,可提高猪诱导性多能干细胞建系效率;已构建的miRNA-205过表达载体将有助于在后续研究中建立稳定表达miRNA-205的细胞系,为进一步深入研究miRNA-205在干细胞重编程和多能性维持中的作用机制打下基础。

Objective：To construct a miRNA-205 overexpression vector and study the effect of miRNA-205 on the establishment of porcine induced pluripotent stem cell lines（i PSCs）. Methods：We compared miRNA transcriptional profiles between the primed porcine embryonic stem cells（primed PESCs）,porcine inner cell mass（ICM）and pig fetal fibroblast cells（PEF）. The miRNA-205 precursor sequence was amplified by PCR and integrated into the expression vector p CAGDNA3-h Fat1 to construct the overexpression vector p CAG-miR205. The expression vector was transfected into porcine fetal fibroblasts containing four mouse derived transcription factors（Oct4,Sox2,Klf4 and c-Myc）by lipofectamine transfection technique. To establish porcine i PSCs,the cells were incubated and cultured in stem cell culture medium. q PCR（real-time quantitative PCR）was used to detect the expression of miRNA-205 in the cells before and after transfection. The inducible effect of miRNA-205 on porcine i PSCs formation was analyzed. Results：The recombinant plasmid p CAG-miR205 was constructed. Compared with untransfected cells,the expression level of miRNA-205 in the transfected cells was significantly increased（P 0.01）,and the colony number was higher after being induced in stem cell culture medium.Conclusion：Over-expression of miRNA-205 at the cellular level can improve the efficiency of porcine induced pluripotent stem cell lineage. The miRNA-205 over-expression vector successfully constructed in this study will help to establish a stable cell line expressing miRNA-205,Which laid the foundation for further study on the exact function and mechanism of miRNA-205 on stem cell reprogramming and pluripotency maintenance.