| Follicular inhibin(follistatin,FST) is a single-chain glycoprotein, which is isolated from the follicular fluid, and also named sex hormone suppressor protein. FST has many biological functions, FST can combine MSTN to form dimmer that can inhibit the binding of MSTN and receptor, Thereby regulate the growth and development of muscle. In order to study the mechanism of effect of FST gene on animal skeletal muscle cell growth, in this study, FST gene was specificity high expressed in skeletal muscle, to investigate the impact of over-expressed FST on growth and development of sheep muscle-derived cells and the expression of related genes. Firstly, liposome mediated method was used to transfect the skeletal muscle-specific expression of FST eukaryotic expression vector PCFCDs-red and PSPFCDs-red respectively into skeletal muscle satellite cells and muscle tube cells, obtaining transgenic cell lines with specific expression of target genes, and then analyze growth status of transgenic cells.Through flow cytometry detection, Real-time quantitative PCR and Western blot methods were used to identify the expression level of target genes and the related gene, and then obtain highly efficient and stable transgenic cell lines, providing a cell model for the preparation of transgenic animals.1. Muscle-specific expression enkaryotic vectors transfected sheep skeletc muscle satellite cells (SMSCs), and then induced muscle tube.Use liposome mediated approach to transfer skeletal muscle-specific vector into sheep skeletc muscle satellite cells, and use G418and red fluorescent protein double selection markers to get positive clones of genetically modified cells. Expand the cultivation of transgenic cells, and obtain transgenic myotube cells through vitro culture. Select positive transgenic muscle satellite cells and myotubes cell, then draw their growth curve. And analyze the cell cycle and cell size of transgenic-positive cells. The results show that the growth trend of selected transgenic cells are basically normal, and similar with the trend of transgenic cells; The growth rate of transgenic skeletal muscle satellite cells is higher than transgenic fetal fibroblast cells; Flow cytometry analysis showed that more than75%cells that have expression vector are in the state of G0/G1, have a single peak and a good aneuploidy.2. The impact of the FST gene on the expression of developmental related genes in sheep muscle-derived cells.The expression of target genes and downstream genes of transgenic cells obtained were detected respectively by real-time quantitative PCR and Western Blotting methods. The results show that expression levels of FST mRNA and protein of transgenic skeletal muscle cells and transgenic myotube cells are higher than untransformed cell lines. In transgenic fetal fibroblast cells, the expression levels of FST have no change. That confirmed the eukaryotic expression vector that containing the skeletal muscle-specific promoter SP and a-actin that we constructed can effectively start FST target gene. And they have a higher expression level in skeletal muscle satellite cells and myotube cells. Among them, the expression level of the FST in transgenic cells that tansfected PSPFCDs-red is higher than that of transfect PCFCDs-red cell lines. This shows that SP promoter has higher start efficiency than a-actin.In the research of genes related muscle development, it was found that in myogenic cells, FST raised can cause the expression levels of MSTN gene increase in RNA transcription, but MSTN protein levels did not change significantly. FST overexpression can cause change of the RNA transcription levels of IGF-I,GH and RHEB genes in sheep muscle-derived cells. |
PDF Full Text Request