Transgenic mice with overexpression of mutated human optineurin(E50K) in the retina
Transgenic mice with overexpression of mutated human optineurin(E50K) in the retina
复制标题
视网膜中过度表达突变人 optineurin (E50K) 的转基因小鼠
DOI：
10.1007/s11033-011-0840-0
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发表时间：
2012-02-01
影响因子：
2.8
通讯作者：
Cheng, Fang
中科院分区：
文献类型：
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作者：
Meng, Qingfeng;Xiao, Zheng;Cheng, Fang
在本工作中，通过PCR使用质粒pBluescript-OPTN实现了插入Glu 50 Lys氨基酸取代的定点突变。构建了突变的人OPTN（E50 K）基因驱动的小鼠c-kit启动子，并经酶切和序列分析证实。通过显微注射方法产生转基因小鼠。用PCR和DNA斑点杂交法筛选阳性转基因小鼠。RT-PCR分析转基因小鼠中突变的人OPTN（E50 K）mRNA表达的RNA水平和定位。采用Western blot和免疫组织化学方法检测突变型OPTN（E50 K）在转基因小鼠中的表达水平和定位。成功建立了人突变型OPTN（E50 K）在视网膜过表达的转基因小鼠模型。将转基因整合并传递到转基因小鼠的染色体中。突变的人OPTN（E50 K）基因由c-kit启动子控制并在小鼠视网膜中表达。在转基因小鼠中突变的人OPTN（E50 K）高于野生型C57 BL/6 J小鼠。我们的研究为研究突变的人OPTN（E50 K）的分子特性提供了一种新的转基因模型。
In the present work, Site-directed mutagenesis to insert the Glu50Lys amino acid substitution was achieved by PCR using plasmid pBluescript-OPTN. Mutated human OPTN(E50K) gene-driven mouse c-kit promoter was constructed and confirmed by endonuclease digestion and sequence analysis. Transgenic mice were generated via the microinjection method. PCR and DNA dot blot were used to screen the positive transgenic mice. RT-PCR analyzed the RNA level and location of mutated human OPTN(E50K) mRNA expression in transgenic mice. Western blot and immunohistochemistry were used to detect the level and location of mutated human OPTN(E50K) expression in transgenic mice. A transgenic mouse model with overexpression of mutated human OPTN(E50K) in retina was successfully established. The transgene was integrated and transmitted into the chromosome of transgenic mice. Mutated human OPTN(E50K) gene was controlled by c-kit promoter and expressed in the retina in mice. Mutated human OPTN(E50K) in transgenic mice was higher than that of wild type C57BL/6J mice. Our studies had provided a new transgenic model for investigating the molecular properties of mutated human OPTN(E50K).