南方农业学报2023，Vol.54Issue(8)：2444-2453,10.DOI:10.3969/j.issn.2095-1191.2023.08.026
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
摘要
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分类
农业科技引用本文复制引用
杨磊,朱正,王博永,梁谦学,吴文德,李恭贺,郑喜邦..Split-GFP双分子荧光互补技术在鸡MSTN基因RNAi检测中的应用[J].南方农业学报,2023,54(8):2444-2453,10.基金项目
国家自然科学基金项目(32060738,31660653) （32060738,31660653）
广西自然科学基金重点项目(2018GXNSFDA281026) （2018GXNSFDA281026）
广西重点研发计划项目(桂科AB16380098) National Natural Science Foundation of China(32060738,31660653) （桂科AB16380098）
Guangxi Natural Science Foundation(2018GXNSFDA281026) （2018GXNSFDA281026）
Guangxi Key Research and Development Plan Project(Guike AB16380098) （Guike AB16380098）
