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

摘要

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 m GH short hairpin RNA( shRNA) was designed according to mouse( Mus musculus) GH mRNA sequence; p Single-t TS-m GH shRNA-RFP,an integrated controllable expression vector of m GH shRNA,was constructed successfully.The recombinant vector was transfected into mouse pituitary tumor cell line At T-20.After addition of doxycyclin( DOX),the expression of red fluorescent protein( RFP) was observed under a fluorescent microscope.The expression level of m GH 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 0.05).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 autocrine / paracrine interactions and analyze functions of GH gene in growth,development and disease occurrence of animals by regulating GH expression levels.