视蛋白基因启动子-绿色荧光蛋白融合基因转基因小鼠模型的建立  

Establishment of transgenic mice harboring mouse rod opsin promoter and enhanced green fluorescent protein fusion gene

作　　者：姚玉成[1] 訾晓渊[1] 李建秀[1] 熊俊[1] 李文林[1] 金艳花[1] 苏小平[1] 倪文君[1] 胡以平[1] 李振林[2] 安靓[2] 杨俊峰 罗清礼 张文 周久模 

机构地区：[1]第二军医大学基础医学部细胞生物学教研室,上海200433 [2]第一军医大学组织胚胎学教研室,广州510515 [3]大学华西医院眼科,四川成都610041

出　　处：《第二军医大学学报》2002年第11期1188-1191,共4页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金资助项目(39800042).

摘　　要：目的:从小鼠基因组中克隆小鼠视蛋白基因启动子(ROP)并建立ROP-绿色荧光蛋白(GFP)融合基因转基因小鼠模型。方法:用PCR法从基因组内扩增ROP,通过基因重组的方法构建pmROP-EGFP表达载体,并用限制性内切酶消化和DNA测序进行鉴定。纯化的pmROP-EGFP表达质粒经Nol I酶切线性化后,用原核显微注射的方法将其注射入小鼠受精卵雄原核,制作转基因小鼠。新生鼠通过PCR检测在基因组水平筛选首建者小鼠,基因组表达阳性的小鼠与正常小鼠交配传代后,取眼球进行冰冻切片,在荧光显微镜下观察视网膜上GFP的表达。结果:从基因组内扩增出了2.1 kb的小鼠ROP,成功构建了小鼠ROP-GFP融合基因表达载体pmROP-EGFP。获得了3只转基因小鼠首建者,分别命名为C57-TgN(mROP-EGFP)SMMU21、C57-TgN(mROP-EGFP)SMMU26、C57-TgN(mROP-EGFP)SMMU27。结论:成功建立了视网膜表达GFP蛋白的C57-TgN(mROP-EGFP)SMMU转基因小鼠,它们可用于研究脑和视网膜发育以及视网膜病变机制,还可为进行视网膜移植实验提供哺乳类动物模型。Objective: To clone mouse rod opsin promoter (ROP) and establish transgenic mice harboring mouse rod opsin promoter and enhanced green fluorescent protein(mROP-EGFP) fusion gene. Methods: Mouse ROP was cloned from C57BL/6 mouse genomic DNA by polymerase chain reaction (PCR). Expression vector of mROP-EGFP fusion gene were constructed by recombination DNA technique. It was identified by restriction endonucleases digestion and confirmed by DNA sequencing. After Not I restriction endonuclease digestion, the coding elements were microinjected into male pronuclei of mice zygotes to generate transgenic mice. The pups were evaluated by PCR at genomic DNA level and mated with normal mouse. Expression of GFP in retina of transgenic mice was detected by fluorescent microscope. Results: 2. 1 kb mouse rod opsin promoter fragment was amplified from mice genome DNA. Expression vector pmROP-EGFP was constructed successfully. Following microinjection of coding sequence of pmROP-EGFP, 3 pups were verified to integrate the mROP-EGFP fusion gene in their genomic DNA by PCR assay, named C57-TgN (mROP-EGFP )SMMU21, C57-TgN (mROP-EGFP)SM-MU26 and C57-TgN(mROP-EGFP) SMMU27. They could express GFP in retina. Conclusion: 2. 1 kb mouse rod opsin promoter is cloned and expression vector pmROP-EGFP is constructed. mROP-EGFP fusion gene transgenic mice are established, which harboring mROP-EGFP gene and expressing GFP in their retina. This is valuable for studying the development of brain and retina, pathogenesis of retina disorder and retina transplanting.

关 键 词：转基因小鼠 视蛋白基因启动子 绿色荧光蛋白 

分 类 号：R373.21[医药卫生—病原生物学]