| MSTN gene is an important negative regulatory factor of muscle growth and development, is one of the most classic genes affecting the body phenotype development. The increment of its expression is closely related to the decrease of muscle mass. Animals could present double-muscle phenotype, if the function of MSTN gene is suppressed. It is of great significant for animal husbandry production, animal genetics and breeding, atrophic muscle disease diagnosis, atrophic muscle disease treatment and atrophic muscle disease prognosis, but MSTN gene could express in the early of embryo. The silence of MSTN gene or the knockout of MSTN gene in embryo period that could cause female animal miscarriage and stillbirth due to the excessive muscle hypertrophy during childbirth. The controllable expressed of MSTN gene becomes the key to realize MSTN gene application value.The study is aim to construct controllable expressed MSTN shRNA vector, prepare to inducible expressed transgenic mice, study the effects of MSTN gene on muscle growth and development by controllable expressed transgenic mice model.We hope it will lay the technological foundation and provide valuable reference for the genetic engineering breeding, muscle atrophy disease drug treatment. The main research contents and results are as follows:Studyl:To construct expressed MSTN shRNA vector and to detect the gene silencing efficiency.In this study,4 pairs of MSTN shRNA were designed based on MSTN mRNA sequence and 4 MSTN shRNA overexpression vectors pGPU6/GFP/Neo-MSTN shRNAs were constructed. After the shRNA interference, the MSTN mRNA and protein in the cell were detected with QRT-PCR and western blot methods. The results showed that 4 MSTN shRNA overexpression vectors have been constructed successfully; they all can reduce the MSTN expression. The cells transfected with pGPU6/GFP/Neo-MSTN714 showed a higher gene silencing efficiency, MSTN mRNA and protein expression level reduced 72% and 74% separately, the difference between them is extremely obvious (P<0.01)Study2:To construct controllable expressed MSTN shRNA vector and to detect the gene inducible expressed efficiency.Selecting the most efficient shRNA MSTN714 to construct controllable expressed vector ptTS-MSTN shRNA. The recombinant vector was transfected to C2C12 cell lines and screened with G418. MSTN shRNA expression was induced with DOX. The GFP expression was observed under a fluorescent microscope and the MSTN mRNA and protein were detected with QRT-PCR and western blot methods. The results showed that, tTS-MSTN shRNA group and the control group without adding DOX, only a small amount of fluorescent expression, after adding DOX, GFP protein signal significantly enhanced; after DOX inducing, the GFP can be observed and the MSTN mRNA decreased 70% in the cells groups transfected with pSingle-tTS-MSTN shRNA-EGFP vector cell groups, the difference between them is extremely obvious (P<0.01).The MSTN protein in this group decreased 90% or so, whereas the other 3 groups showed no obvious difference (P>0.05)Study3:To prepare controllable expressed MSTN shRNA transgenic mice and to evaluate the growth and the development of the mice.Linearizating the tTS-MSTN shRNA carrier, preparating MSTN shRNA controllable expression of transgenic mice with micro injection technology. Testing integrated efficiency of transgenic mice of F0 generation with PCR technology. Detecting the expression of foreign tTS gene of transgenic mice with QRT-PCR technology.The results show that acquiring 8 positive mice from the preparated 159 F0 generation mice.Selecting the best expression efficiency of tTS gene of transgenic mice offspring to evaluate the growth status, reproductive performance, muscular histological performance of transgenic mice. The results showed that the weight of Tg+DOX group mice induced by DOX significantly below Tg, WT+DOX, WT group mice; transgenic mice without DOX inducing, the litter size (7±0.63 & 7.2±0.84) and nest survival number (6.2±0.75 & 6.4±0.55), is no significant differenct from the same period normal mice (WT) (P> 0.05); To compared with MSTN transgenic mice without DOX inducing e and normal mice, MSTN transgenic mice after DOX inducing, the number and cross-sectional area of muscle fiber increased. |
