Establishment Of GFP11-MSTN Expressing Cell Lines And Preliminaryapplication Of Split-GFP In RNA Interference Of Chicken MSTN Gene

Myostatin（MSTN）,originally known as growth differentiation factor 8（GDF-8）,is a member of the TGF-βsuperfamily.MSTN gene is a negative regulator of muscle growth and development.Knockdown or knockout of MSTN gene has important economic value for breeding new breeds of livestock and poultry with double muscling phenotype.Although GFP has become an excellent label protein,there are problems such as its big size,photobleaching and its interfering with the detection of other labels through its permanent expression.GFP11 tag has some advantages,such as its small size,efficient expression as a fusion protein with its targeting protein in tandem.When GFP11is complemented with GFP1-10,GFP fluorescence is generated.Based on bimolecular fluorescence complementarity technology,this split-GFP can still be used as a visual detection marker,reducing the influence of the big-sized GFP label on its fusion protein and cells.There are two primary purposes of this study:a)to screen HEK 293 lines expressing GFP11-MSTN gene;b)to evaluate RNAi efficiency of MSTN in the cell lines by comparing detection results of split-GFP analysis,q RT-PCR and Western blotting assay,further verifing the effectiveness of split-GFP technique in return.Experiment 1.Screening of GFP11-MSTN expressing cell linesA pl VX-GFP11-MSTN lentivirus vector and a pc DNA3.1（+）-GFP1-10vector were constructed,and the effectiveness of Split-GFP system was verified by co-transfection of HEK 293T cells using both of the plasmids.pl VX-GFP11-MSTN vector was packed and tilted,with which HEK 293T cells was infected to screen puromycin-resistent 293T^GFP11-MSTNcell lines.The expression level of GFP11-MSTN were detected by q RT-PCR,Western Blotting and Split-GFP systems.The results showed that:（1）DNA sequencing indicated that p LVX-GFP11-MSTN and pc DNA3.1（+）-GFP1-10 were constructed successfully;GFP positive（GFP+）cells was not observed post transfection of HEK 293T cells with either pl VX-GFP11-MSTN or pc DNA3.1（+）-GFP1-10,but GFP+cells were seen post transfection with combination of the two plasmids,which preliminatively verified the effectiveness of split-GFP.（2）q RT-PCR and Western Blotting confirmed that 293T^GFP11-MSTNcell lines was obtained,and split-GFP system was capable of reflecting MSTN expression level.Experiment 2.RNAi mediated knockdown of chicken MSTN geneThree sh RNA lentiviral vectors,sh RNA-a,sh RNA-b and sh RNA-c,were constructed in terms of three different targeting sites of MSTN gene.Being screened by q RT-PCR and split-GFP system,sh RNA-a was packaged because of its higher interferential efficiency.The virus particles were tiltled and the optimal MOI for infecting HEK 293T cells was determined.Being infected with sh RNA-a lentivirus,and being screened with Hygromycin B,the expanded culture of 293T^GFP11-MSTNcells was instantaneously transfected with the pc DNA3.1（+）-GFP1-10 vector.The effect of RNA interference was detected by q RT-PCR,Western Blotting and Split-GFP system.The results showed that:（1）three sh RNAs were effective on MSTN knockdown,among which sh RNA-a is the best one,since it targets to exon 1 of MSTN gene.（2）lentivirus vector sh RNA-a was successfully packed,and its titer was 2.85×10⁸TU/m L;its optimal MOI for infecting 293T^GFP11-MSTNcells was 3,and the optimal concentration of Hygromycin B was 250μg/m L.（3）Post knockdown of MSTN by infection of293T^GFP11-MSTNcells with sh RNA-a lentivirus particles,both fluorescence microscopy and flow cytometry showed that the percentage of GFP+cells in the RNA interference group was significantly decreased compared with that in the negative control group.Similar results were obtained by q RT-PCR and Westtern Blotting.The effectiveness of split-GFP system was further verified.In summary,we obtained cell lines that expressing GFP11-MSTN stably,and the effectiveness of Split-GFP system was verified by RNA interference.This study provides an ideal experimental material for the functional study of MSTN gene,and contributes to generating a new breed of broiler with double muscling phenotype through injection of sh RNA-a lentivirus into chicken embryos.