Split-GFP双分子荧光互补技术在鸡MSTN基因RNAi检测中的应用
Application of Split-GFP bimolecular fluorescence complemen-tary technique in detecting RNAi of chicken MSTN gene
杨磊 1朱正 1王博永 1梁谦学 1吴文德 1李恭贺 1郑喜邦1
作者信息
- 1. 广西大学动物科学技术学院,广西南宁 530004
- 折叠
摘要
[目的]以Split-GFP双荧光互补技术检测鸡肌肉生长抑制素基因(MSTN)RNA干涉(RNAi)效果,并与其他常用检测方法比较,以验证Split-GFP双分子荧光互补技术在RNAi效果评估中的有效性和可行性.[方法]将合成的3个shRNA慢病毒载体(shRNA-a、shRNA-b和shRNA-c)分别转染稳定表达GFP11-MSTN融合蛋白的HEK 293TGFP11-MSTN细胞,经实时荧光定量PCR筛选出最佳shRNA慢病毒载体并包装为慢病毒,然后以慢病毒感染HEK 293TGFP11-MSTN细胞,采用潮霉素B筛选mCherry阳性(mCherry+)细胞,再以实时荧光定量PCR和Western blotting检测RNAi效果;得到的mCherry+细胞再转染pcDNA3.1(+)-GFP1-10质粒,通过荧光显微镜观察和流式细胞术评估RNAi效果.[结果]3个shRNA慢病毒载体对MSTN基因表达均有极显著的抑制作用(P<0.01,下同),其中又以Anti-MSTN shRNA-a慢病毒载体的干涉效果最佳.Anti-MSTN shRNA-a慢病毒感染HEK 293TGFP11-MSTN细胞的最适MOI=3,该条件下MSTN基因相对表达量及GFP11-MSTN融合蛋白表达量均受到抑制;得到的mCherry+细胞再转染pcDNA3.1(+)-GFP1-10质粒,荧光显微镜观察和流式细胞术检测结果表明,GFP+细胞数量明显减少,GFP+细胞百分率由31.1%降至11.5%.Split-GFP检测结果与实时荧光定量PCR及Western blotting检测结果相符,说明Anti-MSTN shRNA-a慢病毒能有效抑制GFP11-MSTN融合蛋白表达,发挥了RNAi作用.[结论]以Anti-MSTN shRNA-a慢病毒对MSTN基因的干涉效果最佳,其感染HEK 293TGFP11-MSTN细胞后MSTN基因表达极显著下调,且再转染pcDNA3.1(+)-GFP1-10质粒后细胞中的GFP+细胞百分率明显下降,与实时荧光定量PCR和Western blotting检测结果相符,证实Slipt-GFP双分子荧光互补技术是一种可靠的可视化RNAi检测方法.
Abstract
[Objective]The purpose of this study was to detect the efficacy of RNA interference(RNAi)of chicken myostatin gene(MSTN)by Split-GFP bimolecular fluorescence complementary technique and to compare it with com-monly used detection methods in order to validate the effectiveness and availability of the Split-GFP bimolecular fluores-cence complementary technique in the assessment of RNAi efficacy.[Method]The three synthesized shRNA lentiviral cloning vectors(shRNA-a,shRNA-b and shRNA-c)were respectively transfected to HEK 293TGFP11-MSTN cells which sta-bly expressed the GFP11-MSTN fusion protein.The optimal shRNA lentiviral cloning vector was screened by real-time fluorescence quantitative PCR and packaged as lentivirus.Subsequently,the HEK 293TGFP11-MSTN cells were infected with the lentivirus,and mCherry positive(mCherry+)cells were screened using hygromycin B,and the RNAi efficiency was detected by real-time fluorescence quantitative PCR and Western Blotting.The obtained mCherry+ cells were trans-fected with a pcDNA3.1(+)-GFP1-10 plasmid,and the RNAi efficiency was assessed by fluorescence microscopy obser-vation and flow cytometry.[Result]All of three shRNA lentiviral vectors had extremely significant inhibitory effects on MSTN gene expression(P<0.01,the same below),among which Anti-MSTN shRNA-a lentiviral vector had the best inter-fering efficacy.Anti-MSTN shRNA-a lentivirus infected HEK 293TGFP11-MSTN cells at the optimal MOI=3,and the relative expression of the MSTN gene and the expression of the GFP11-MSTN fusion protein were inhibited under this condition.The obtained mCherry+ cells were re-transfected with pcDNA3.1(+)-GFP1-10 plasmid,and both fluorescence microsco-py observation and flow cytometry assay results showed that the number of GFP+ cells was decreased greatly,and the per-centage of GFP+cells was reduced from 31.1%to 11.5%.The results of Split-GFP assay were consistent with those of real-time fluorescence quantitative PCR and Western Blotting detections,indicating that Anti-MSTN shRNA-a lentivirus could inhibit GFP11-MSTN fusion protein expression,thus playing a RNAi role.[Conclusion]Anti-MSTN shRNA-a lentivirus has the highest interfering efficacy on MSTN gene,which extremely significantly down-regulates the expression of MSTN gene after infection of HEK 293TGFP11-MSTN cells,and the percentage of GFP+ cells in the cells is greatly decreased after re-transfection of pcDNA3.1(+)-GFP1-10 plasmid,which is consistent with the results of real-time fluorescence quantita-tive PCR and Western blotting detection results,confirming that Slipt-GFP bimolecular fluorescence complementary tech-nique is a dependable and visualized method for RNAi detection.
关键词
肌肉生长抑制素(MSTN)/RNA干涉(RNAi)/shRNA/慢病毒/Split-GFP双分子荧光互补技术Key words
myostatin(MSTN)/RNA interference(RNAi)/shRNA/lentivirus/Split-GFPbimolecular fluorescence complementary technique引用本文复制引用
基金项目
国家自然科学基金(32060738)
国家自然科学基金(31660653)
广西自然科学基金重点项目(2018GXNSFDA281026)
广西重点研发计划项目(桂科AB16380098)
出版年
2023
国家科技期刊平台
NSTL
维普
万方数据