增强子驱动的Cre转基因小鼠的构建及Cre基因的表达鉴定  

Expression of Cre Recombinase in an Enhancer-Cre Transgenic Mouse

作　　者：王立良[1] 王淼 张玉建 王浩 刘勇[1] 邵一鸣 

机构地区：[1]中国疾病预防控制中心性病艾滋病预防与控制中心,北京100176 [2]University of Pennsylvania [3]ApoCell Biosciences,Inc. [4]Baylor College of Medicine

出　　处：《生物技术通讯》2010年第3期304-309,共6页Letters in Biotechnology

摘　　要：目的:探索将增强子应用于构建Cre转基因小鼠品系,为以条件基因敲除为基础的基因功能研究提供更多的工具。方法:通过PCR方法从小鼠的细菌人工染色体扩增UH增强子片段,构建含有Hsp68基础启动子、增强子UH、Cre重组酶基因和SV40 polyA的转基因载体pLW400,将3.3 kb的转基因片段通过显微注射导入小鼠受精卵;为了检测Cre在转基因小鼠中的表达,将转基因一代小鼠与纯合子ROSA26报告小鼠(R/R)交配,收集第14 d胚胎期(E14)的舌组织进行LacZ染色检测鉴定。结果:经鉴定,31只子代小鼠中有6只携带外源基因,整合率为19.4%;与R/+对照相比,E14期的双基因型Cre,R/+舌组织为阳性结果(蓝色)。这表明Cre基因在转基因小鼠舌组织内得到表达,并在体内介导ROSA26基因座loxP位点间的重组,且有效删除了2个loxP之间的片段,从而启动了LacZ基因的表达。结论:构建了UH增强子-Hsp68Cre的转基因小鼠,在舌肌中特异表达Cre基因,提示增强子可以被选择应用于Cre转基因小鼠的构建;为舌肌的发育和再生研究奠定了基础。Objective：To develop Cre transgenic mice which expression was driven by enhancer-Hsp68 minimal promoter.Methods：To create Cre transgenic mouse,a transgenic construct（pLW400） was made with the following four elements：an Hsp68 basic promoter,a Cre gene,an enhancer from one mouse bacterial artificial chromosome（BAC） clone and a polyA signal.The 3.3 kb DNA fragment of Cre expression cassette was injected into mouse fertilized eggs to generate transgenic mouse.To check expression pattern of Cre in the transgenic mice,the F1 mice were crossed with homozygous ROSA26 reporter（R / R） mice.Tongues from embryos 14 days（E14） were collect-ed,and whole-mount LacZ staining of embryos（or tongue） was performed.Results：6 out of 31 offspring were iden-tified as founders carrying the Cre transgene,with integration efficiency 19.4%.Compare with tongue tissue from R / ＋ control,the tongue tissue from Cre,R / ＋ bigenic embryo showed positive staining signal（blue color）.This sug-gested Cre gene had been successfully expressed with recombinant activities in the transgenic mice,and it can be used to modify genes of interest.Conclusion：The transgenic lines expressing Cre recombinase driven by enhancer were successfully generated.

关 键 词：增强子 CRE重组酶 转基因小鼠 ROSA26报告小鼠 LacZ染色 

分 类 号：Q789[生物学—分子生物学]