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Optimal screening of shRNA molecules and reconstruction of the shRNA eukaryotic expression vector of GFAP expressive suppression of rat spinal cord-derived astrocytes
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- Author:
- No author available
- Journal Title:
- CHINESE JOURNAL OF NEUROMEDICINE
- Issue:
- 11
- DOI:
- 10.3760/cma.j.issn.1671-8925.2007.11.002
- Key Word:
- 胶质原纤维酸性蛋白;RNA干扰;小干扰RNA
Abstract： 目的 构建并筛选大鼠胶质原纤维酸性蛋白(GFAP)表达抑制短发夹样RNA(shRNA)真核表达载体.方法 针对GFAP基因全编码序列设计并合成三对9 bp茎环结构、19bp干扰序列特异性shRNA模板,体外定向克隆构建特异性重组质粒真核表达载体;通过体外大鼠脊髓源星形胶质细胞GFAP表达抑制模型,脂质体介导RNA干扰分子转染,实时荧光定量RT-PCR及Western blot技术观察RNA干扰后原代星形胶质细胞GFAP表达抑制效果,筛选最佳GFAP表达干扰抑制真核表达载体.结果 序列测定证实GFAP-shRNA重组质粒真核表达载体构建成功,三对shRNA模板在mRNA及蛋白表达水平抑制靶基因表达效率分别为81%、63%、56%.结论 高效率的GFAP-shRNA真核表达载体在大鼠原代星形胶质细胞GFAP表达抑制模型中能高效抑制GFAP基因表达,为后续多靶点RNA干扰技术在脊髓损伤胶质瘢痕抑制基因治疗中的应用奠定了前期基础.
- [1]何祥梁,何东华,黎锋,等.人resistin基因原核表达载体构建和表达[J].2004,(5).
- [2]张燕,薛耀明,郭爱林,等.GST-PPARγC1原核表达载体的构建、表达及抗GST-PPARγC1多克隆抗体的制备[J].2005,(5).
- [3]Thomas, Tuschl.Expanding small RNA interference.[J].2002,20(5).
- [4]Jing NH,Chen YG,Xia CH,etc.Cell aggregation-induced FGF8 elevation is essential for P19 cell neural differentiation[J].2006,17(7).
- [5]Angela, Reynolds,Devin, Leake,Queta, Boese,etc.Rational siRNA design for RNA interference.[J].2004,22(3).
- [6]Kwak YD,Choumkina E,Sugaya K.Amyloid precursor protein is involved in staurosporine induced glial differentiation of neural progenitor cells[J].2006,344(1).