Application of Split-GFP bimolecular fluorescence complementary technique in detecting RNAi of chicken MSTN gene

【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 commonly used detection methods in order to validate the effectiveness and availability of the Split-GFP bimolecular fluorescence complementary technique in the assessment of RNAi efficacy.【Method】The three synthesized shRNA lentivira cloning vectors(shRNA-a, shRNA-b and shRNA-c) were respectively transfected to HEK 293T^GFP11-MSTN cells which stably expressed the GFP11-MSTN fusion protein.The optimal shRNA lentiviral cloning vector was screened by realtime fluorescence quantitative PCR and packaged as lentivirus.Subsequently, the HEK 293T^GFP11-MSTN cells were infected with the lentivirus, and mCherry positive(m Cherry⁺) 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 transfected with a pcDNA3.1(+) -GFP1-10 plasmid, and the RNAi efficiency was assessed by fluorescence microscopy observation 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 interfering efficacy.Anti-MSTN shRNA-a lentivirus infected HEK 293T^GFP11-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 microscopy observation and flow cytometry assay results showed that the number of GFP⁺ cells was decreased greatly, and the percentage of GFP⁺ cells was reduced from 31.1%to 11.5%.The results of Split-GFP assay were consistent with those of realtime 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 293T^GFP11-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 quantitative PCR and Western blotting detection results, confirming that Slipt-GFP bimolecular fluorescence complementary technique is a dependable and visualized method for RNAi detection.