旨在构建整合型可控表达猪生长激素(porcine growth hormone,pGH)基因转基因小鼠模型,通过控制GH的表达时间和表达量来减少 GH过早表达产生的副作用,对于培育高饲料转化率大家畜具有重要研究意义.利用本课题组前期成功构建并在PK15细胞系中验证的整合型诱导表达 pGH四环素(Tet-on)载体,通过原核显微注射方法制备转基因小鼠.以Southern blot鉴定为阳性的 Founder鼠为基础扩繁培育,对转基因后代鼠3～10周龄通过饮水中添加诱导底物强力霉素(DOX)进行诱导试验;利用RT-qPCR技术检测外源基因表达水平;利用放射性免疫检测方法对小鼠血液中猪生长激素水平进行定量检测,同时称重,评估转入基因对小鼠机体表型的影响.结果表明,转基因小鼠诱导组的体重与转基因小鼠非诱导组和同窝非转基因组小鼠相比显著增大(P＜0.05),血清中pGH水平显著上升(P＜0.05),说明外源GH有效表达并且调节动物机体的生长.结果提示,该模型的建立有助于深入研究 GH对机体发育的影响以及机体中影响GH 分泌的分子机制和信号通路,促进GH在家畜生产中的应用,也为制备安全、高效的GH转基因大家畜奠定理论基础.%This study aimed to construct an integrated and controllable transgenic mice model with pGH gene expression,which could reduce the side effects caused by premature expression of GH through controlling the time and quantity of GH gene expression,it would become a meaningful research to improve feed conversion of livestock.The transgenic mice were successfully obtained by prokaryotic microinjection method using a vector of Tet-on regulatory expression system,which was constructed successfully and verified through transfected PK15 cell line.Based on Southern blot results,positive Founders were bred for getting offspring.When we got the trans-genic offspring,the induction experiment was carried out with the induction substrate doxycyc-line(DOX)supplementation in the drinking water of the transgenic mice at 3-10 weeks old.RT-qPCR technology was used to detect the level of exogenous genes expression.The radioactive im-munoassay method was used to detect porcine growth hormone content in serum of mice.Body weight was weighed to evaluate the effects of pGH gene on phenotype of transgenic mice.The results showed that the body weight of induced transgenic mice was higher than that of non-induced transgenic mice and non transgenic mice in the same litter(P＜0.05);The level of pGH in serum of transgenic mice was significant higher than that of mice in control(P＜0.05),which indicated that exogenous GH could effectively express and regulate the growth of mice body.The establishment of model of pGH transgenic mice contributed to explore the effect of GH gene on body development and the molecular mechanism and signal pathway of growth hormone secre-tion.It would promote the application of GH in livestock production ;it also provided a theoreti-cal foundation and technical support for the preparation of safe and efficient transgenic livestock with GH gene.
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