摘要
目的:建立带有人类骨保护素OPG基因启动子驱动报告基因LacZ的转基因小鼠模型,为OPG体内转录水平的表达调控研究和药物筛选创造条件。方法:将克隆到的人类OPG基因5′端上游6.0kb非翻译序列作为启动子,大肠杆菌编码β半乳糖苷酶的LacZ基因作为报告基因,构建表达载体pCINeoOPGLacZ。经显微操作注射到受精卵原核中,经PCR以及Southern印迹杂交鉴定转基因阳性小鼠;用RTPCR分析LacZ在组织中的表达;利用邻硝基苯βD半乳吡喃糖苷(ONPG)作为底物反应后比色分析组织中的β半乳糖苷酶活性。结果:构建完成的表达载体pCINeoOPGLacZ质粒经酶切和测序鉴定序列正确,线性化后显微注射。PCR以及Southern印迹杂交鉴定获得了10只转基因小鼠(Founders),经交配繁育,建立了5个转基因小鼠系,RTPCR分析表明其中一个系小鼠组织中表达LacZ基因,与内源OPG表达模式一致,组织中可以广泛检测到相应的β半乳糖苷酶活性。结论:成功建立了人类OPG基因启动子驱动报告基因LacZ的转基因小鼠,为体内研究OPG转录水平的表达及药物筛选提供了理想的动物模型。
Objective： To generate transgenic mouse model for studying the transcriptional regulation of human osteoprotegerin（OPG） and for new drug screening in vivo. Methods ： The human 6.0kb OPG promoter and the E. coli LacZ gene（3.3kb） were obtained by PCR. Then the expression vector pCI-Neo-OPG-LacZ was constructed by inserting OPG promoter and LacZ gene into the pCI-Neo-based plasmid. The plasmid was linearized and microinjected into fertilized eggs according to standard protocol. Transgenic founders were detected by PCR and Southern blot. LacZ gene expression and β-galactosidase enzyme activity were analyzed by RT-PCR and ONPG reaction. Results： Five transgenic mouse lines have been generated. RT-PCR analysis showed that LacZ gene expression was detected in 1 out of 5 transgenic lines. The expression pattern was identical to the endogenous OPG gene. The activity of β-galactosidase could be detected in corresponding tissue homogenates. Conclusion： A transgenic mouse model was generated which is useful for studying transcriptional regulation of human OPG and for new drug screening in vivo.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2005年第11期12-15,共4页
China Biotechnology
基金
国家"863"计划资助项目(2004AA216080)
上海市科技发展基金资助项目(99JC14029
99XD14005)
作者简介
通讯作者，电子信箱：zhugangw@shsmu．edu．cn
