Construction of Controllable Expression Vector of Mus musculus Growth Hormone (GH) shRNA and Its Gene Si- lencing Efficiency Detection

Growth hormone （GH） is a polypeptide hormone secreted by the pituitary, which promotes animal growth, muscle development, metabolism regulation and other important physiological functions. In this study, a pair of mGH short hairpin RNA （shRNA） was designed according to mouse （ Mus musculus） GH mRNA sequence; pSingle-tTS-mGH shRNA-RFP, an integrated controllable expression vector of mGH shRNA, was constructed successfully. The recombinant vector was transfected into mouse pituitary tumor cell line AtT-20. After addition of doxycyelin （ DOX）, the expression of red fluorescent protein （RFP） was observed un- der a fluorescent microscope. The expression level of mGH in cells was detected by quantitative Realtime PCR （qRT-PCR） and Western blot. After DOX induction, the relative expression level of GH mRNA in cells transfected with GH shRNA was reduced by about 70% compared with that in DOX-free group and other control groups, exhibiting extremely significant differences （P 〈 0.01 ） ; moreover, the relative expression level of GH protein was reduced by about 90% ; the expression level of GH mRNA and GH protein exhibited no significant difference among other groups （P 〉 0.05）. In this study, a controllable expression vector of GH shRNA with high gene silencing efficiency was constructed successfully, which could be used to reveal GH autocfine / paracrine interactions and analyze functions of GH gene in growth, development and disease occurrence of animals by regulating GH expression levels.

Application of Real-time Fluorescent Quantitative PCR in Studies on Plants

Real-Lime fluorescent quantitative PCR is a method for quantitative analysis of gene expression developed in recent years, which has been widely used in various fields such as basic scientific research, clinical diagnosis, disease study, drug research and development since its appearance. It starts relatively late in study on plants, but has already been used for analysis of gene expression in plants and gene identification of exogenous genes. The principles or advantages and dis- advantages of real-time fluorescent quantitative PCR, or its potential problems and condition optimizations in tests were introduced in this study, and then the appli- cation and prospect of real-time fluorescent quantitative PCR in study on plants were also been discussed.