Kap147/Kpn杂合启动子引导Cre重组酶在转基因小鼠睾丸组织中特异性表达  

A Testis-Specific Expression of Cre Recombinase in Transgenic Mice Testis by Kap147/Kpn Hybrid Promoter

作　　者：刘飞[1] 孟军[1] 范立强[1] LIU Fei;MENG Jun;FAN Liqiang(State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China)

机构地区：[1]华东理工大学生物反应器工程国家重点实验室

出　　处：《华东理工大学学报（自然科学版）》2019年第6期919-927,共9页Journal of East China University of Science and Technology

基　　金：国家自然科学基金（81071252）

摘　　要：为了验证杂合启动子Kap147/Kpn的体内、外功能,构建了Kap147/Kpn引导的Cre重组酶转基因鼠。体外细胞转染和激素诱导证实Kap147/Kpn启动子具有细胞特异性,且受雄激素诱导。通过RT-PCR(Reverse Transcription-Polymerase Chain Reaction)和荧光定量PCR(Polymerase Chain Reaction)证实Kap147/Kpn启动子引导Cre重组酶在转基因鼠(Cre-F0-2)睾丸组织中特异表达。荧光定量PCR检测发现,Cre mRNA在转基因鼠睾丸中的表达量与小鼠年龄相关,经15 mg/(kg·d)二氢睾酮(DHT)处理后睾丸中Cre mRNA表达量增加1.7倍,经100 mg/(kg·d)醋酸环丙孕酮(CAP)处理后Cre mRNA表达量降低约50%,证实Kap147/Kpn启动子在转基因鼠体内受雄激素调控。荧光组化显示,转基因鼠Cre-F0-2与双荧光Cre重组酶报告鼠杂交后,能成功介导子代鼠睾丸组织特异性的基因敲除。杂合启动子Kap147/Kpn引导的Cre重组酶转基因鼠(Cre-F0-2)具有雄激素调节性和睾丸靶向性,是一个潜在的研究睾丸基因功能的有力工具。Gene targeting technology has been widely applied to gene function analysis. In order to precisely study gene function in specific tissues during a specific developmental period, tissue specific or inducible promoter driven Cre recombinase to guide gene knockout or silence are often used. To study the in vitro and in vivo functions of the hybrid promoter Kap147/Kpn and to facilitate the study of gene function using Cre/loxp system, the transgenic mice of Cre recombinase guided by the heterozygous promoter Kap147/Kpn were constructed. Co-transfection assay, in vitro cell culture experiments and dihydrotestosterone induction confirmed Kap147/Kpn promoter is cell specific and androgen inducible. Reverse transcription PCR and fluorescence quantitative PCR confirmed the testis-specific Cre gene expression in the offspring Cre-F0-2 and kidney-specific Cre gene expression in the offsprings of Cre-F0-1 and Cre-F0-6. In addition, fluorescence quantitative PCR assay showed that the amount of Cre mRNA in adult mice of CreF0-2 offsprings was 20-30 times higher than that in the newborn or senile mice, which is in accordance with the amount of androgen in mice, suggesting that the activity of Kap147/Kpn chimeric promoter is probably regulated by androgen. Hormone induction experiment showed that 15 mg/(kg·d) DHT accelerated the expression of Cre mRNA of about 1.7-fold in testis, and 100 mg/(kg·d) CAP reduced the expression by about 50%, which thereby demonstrated that the Kap147/Kpn hybrid promoter was androgen inducible in Cre-F0-2 transgenic mice. Upon breeding the Kap147/Kpn-Cre mice(Cre-F0-2) with double-fluorescent Cre reporter mouse(B6.129(Cg)-Gt(ROSA) 26 Sor), frozen sections from male double transgenic mice showed bright green fluorescence only in testis where Cre recombinase specifically expressed. This model mouse Cre-F0-2 with tissue specific Cre recombinase in the testis would provide a novel useful tool for studying the physiological significance of genes expressed in the testis.

关 键 词：基因打靶 肾脏雄激素调节蛋白 Cre转基因鼠 睾丸 

分 类 号：Q291[生物学—细胞生物学]