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Transfection and initial detection of human GAD67 gene into bone marrow stromal cells-derived neural stem cells
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- Author:
- No author available
- Journal Title:
- CHINESE JOURNAL OF NEUROMEDICINE
- Issue:
- 3
- DOI:
- 10.3760/cma.j.issn.1671-8925.2007.03.004
- Key Word:
- 谷氨酸脱羧酶67;转染;骨髓基质细胞;神经干细胞
Abstract： 目的 利用转基因技术将人类谷氨酸脱羧酶(GAD)67基因转染骨髓基质源性神经干细胞(BMSCs-D-NSCs),获得GAD67修饰的成体专能干细胞.方法 以PCR、双酶切及测序鉴定pEGFP-C2-GAD67和pcDNA3.1-GAD67重组子;利用密度梯度离心法分离培养大鼠BMSCs并促其向NSCs方向分化,将编码人类GAD67基因的真核表达质粒pEGFP-C2-GAD67及pcDNA3.1-GAD67分别转染BMSCs-D-NSCs,荧光显微镜下观察并用流式细胞仪检测转染效率. 结果 pEGFP-C2-GAD67及pcDNA3.1-GAD67均可扩增出约1 800bp目的条带,经双酶切和基因测序证实.荧光显微镜下观察可见大量绿色荧光细胞,两种载体转染效率分别为39.3%和40.5%.结论 pEGFP-C2-GAD67及pcDNA3.1-GAD67真核表达载体重组正确,利用脂质体介导人类GAD67基因能有效转染BMSCs-D-NSCs,为进一步观察其GAD67基因和蛋白的表达提供了前提条件.
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