RNAi In Porcine MyoD And Myostatin Gene And The Relationship Between The Gene Functions

The quality and quantity of the skeletal muscle are important economy characters in porkproduction. Skeletal muscle growth is a very complicated process which requires the regulationfrom both the myogenic family and the myostatin gene. MyoD is one of the myogenic regulatoryfactors, and Myostatin is a negative regulator of myogenesis. Researches on the function ofMyoD and Myostatin gene will make a great sense on pig breeding and the understanding of themuscle generation mechanism.With the successful researches on mammalian gene mapping, genomics studies are focusingtheir researches on elucidating the function of genes. RNA interference (RNAi) has been shownto be a powerful method and a frontier technology to study the function of genes by silencingendogenous mRNA with double-stranded (ds) RNA. The following studies analyzed the changesin gene expression and cell proliferation before and after the MyoD and Myostatin gene RNAinterference in porcine skeletal muscle satellite cell. To analyze the relationship between MyoDand Myostatin gene, one is downregulated when the other is being studied. The results are asfollows:Cultural system for porcine skeletal muscle satellite cell in vitro was established. The cellswere identified by karyotype analysis and morphology method.The level of MyoD and Myostatin gene expression were analyzed by RT-PCR in fivegenerations. The expression level of MyoD were 0.432, 0.455, 0.352, 0.319, 0.289; theexpression level of Myostatin were 0.414, 0.461, 0.318, 0.319, 0.285. High level expressionsof both MyoD and Myostatin were observed in the second generation. There was significantdifference between the second generation and other generation (P＜0.05). Expression changes ofMyostatin are in accordance with MyoD.Three pairs of specific siRNAs were designed in different point of MyoD mRNA.Recombinant plasmid constructed by psiSTRIKE vector was used to transfect porcine skeletalmuscle satellite cell. MyoD gene was silenced in porcine skeletal muscle satellite cellsuccessfully. The best interference system for MyoD and Myostatin gene was screened and confirmed.Using different concentrations (0μg/ml. 100μg/ml, 200μg/ml, 300μg/ml,400μg/ml,500μg/ml,600μg/ml,700μg/ml and 800μg/ml ) to screen G418, 200μg/ml was the bestmaintaining concentration and 400μg/ml was the best screening concentration. After affected bythe three pair of specific siRNAs, the downregulation of MyoD genes were 0.098, 0.118, 0.188separately. The third pair of specific siRNAs can significantly inhibit the MyoD mRNAexpression (P＜0.05). Analyzing the mRNA expression after RNA interference after 12,22 and32 days, the mRNA expression of MyoD were 0.188, 0.165, 0.144, the mRNA expression ofMyostatin gene were 0.199,0.097,0.073. The expression level of these two genes were bothsignificantly downregnlated in 12days (P＜0.05).The influences of MyoD and Myostatin gene interference on porcine skeletal musclesatellite cell proliferation were studied. Interference of MyoD gene had no effect on cellproliferation (P＞0.05).Interference of Myostatin can significantly increase cell proliferation capacity (P＜0.05).Changes of MyoD and Myostatin gene expression after gene interference in porcine skeletalmuscle satellite cell were studied. The expression of Myostation gene was significantly declinedafter the downregulation of MyoD gene (P＜0.05). The expression of MyoD gene wassignificantly increased after the downregulation of Myostation gene (P＜0.05).In all, after the MyoD and Myostatin gene RNA interference the proliferation of porcineskeletal muscle satellite cell are related to these two genes expressions.