Silence Of PID1Gene Expression Using RNA Interference In C2C12Cell Line

With the improvement of the living standard, consumers and producers pay moreattention to the problem on pork quality. Intramuscular fat（IMF）content is an importantquality indicator of measuring pork, it has a remarkable positive effect in meat tenderness,juiciness and flavor. Currently, the regulation mechanism of porcine intramuscular fatdeposition is not clear, the major gene has not yet found. The PID1play an important role infat cell proliferation, was highly expressed in obese people. We are laboratory studies thePID1gene in Laiwu pigs, Lulai Black pigs and Yorkshire, in backfat, longissimus muscle andliver in differential expression of and relationship with the IMF. The results implied that theexpression of PID1may be related to fat deposition. In this study, by the way of building amouse model, by building the small short hairpin ring interfering RNA expression vector forthe PID1, it’s under the control of the U6promoter, the interference vector was transfectedinto C2C12cells by RT-PCR and Western blot analysis of shRNA vectors on C2C12cellsdownward effect in the process PID1mRNA and protein expression, to lay the foundation forfurther study the function of the the verification PID1gene IMF deposition.To construct short hairpin RNA (shRNA) expression vector that can interference PID1expression in mouse. Four short hairpin RNA sequences and the negative interferingsequences were designed through mouse PID1cDNA sequence. The correspondingdouble-stranded DNA sequences were used to construct pGPU6/GFP/Neo vector, namelypGPU6/GFP/Neo-shRNA-A, pGPU6/GFP/Neo-shR NA-B, pGPU6/GFP/Neo-shRNA-C,pGPU6/GFP/Neo-shRNA-D and pGPU6/GFP/N eo-shRNA-NC. They were transfected intothe C2C12cells, in which the silencing effect on PID1expression was investigated byRT-PCR and Western blot. The expression vector targeting on PID1were successfullyconstructed, and confirmed by agarose electrophoresis and sequence analysis. The fourexpression vectors were transfected into the C2C12cells, after24h, the PID1mRNAexpression was decreased by (23.58±1.87)%,(75.44±0.77)%,(70.52±0.41)%and(56.60±3.13)%, respectively, and after48h, its protein expression was decreased by(30.15±5.05)%,(71.86±4.85)%,(67.93±2.28)%and (56.81±2.01)%, correspondingly. Theconstructed expression vector pGPU6/GFP/Neo-shRNA-B, pGPU6/GFP/Neo-shRNA-C andpGPU6/GFP/Neo-shRNA-D can effectively inhibit the expression of PID1mRNA and protein, which could provide a basis for further research on the function and mechanism ofPID1.