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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 opsin gene promoter (ROP) from mouse genome and establish transgenic mouse model of ROP-GFP fusion gene. METHODS: ROP was amplified from the genome by PCR, and pmROP-EGFP expression vector was constructed by gene recombination. The recombinant plasmid was identified by restriction endonuclease digestion and DNA sequencing. The purified pmROP-EGFP expression plasmid was linearized by Nol I digestion and then injected into the pronucleus of mouse fertilized egg by pronuclear microinjection to make transgenic mice. Neonatal rats were screened by PCR for first generation mice at genomic level. Mice with positive genomic expression were passaged with normal mice after subculture. Frozen sections were taken from the eyeballs and the expression of GFP on the retina was observed under a fluorescence microscope. Results: A 2.1 kb mouse ROP was amplified from the genome. The murine ROP-GFP fusion gene expression vector pmROP-EGFP was successfully constructed. Three transgenic mice were named as C57-TgN (mROP-EGFP) SMMU21, C57-TgN (mROP-EGFP) SMMU26 and C57-TgN (mROP-EGFP) SMMU27 respectively. CONCLUSIONS: C57-TgN (mROP-EGFP) SMMU transgenic mice with GFP protein expression on the retina were successfully established. They can be used to study the mechanisms of brain and retina development and retinopathy. Mammalian models can also be provided for retinal transplantation experiments.