Gene transfer methods for CNS organotypic cultures: A comparison of three nonviral methods
Gene transfer methods for CNS organotypic cultures: A comparison of three nonviral methods
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DOI：
10.1006/mthe.2000.0235
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发表时间：
2001-01-01
影响因子：
12.4
通讯作者：
Messer, A
中科院分区：
文献类型：
--
作者：
Murphy, RC;Messer, A
从出生后第12天的小鼠小脑组织切片培养物中，用三种非病毒方法：生物枪法、脂质体转染法和电穿孔法进行基因转染法。重组表达载体pHD17-25Q-GFP编码含有绿色荧光蛋白(GFP)的融合蛋白。脂肪转移和电穿孔的最佳条件与以前在活体动物模型中发现的条件相同。在切片培养中，电穿孔(26+/-6)和生物化学(34+/-4.4)提供了比脂质体(15+/-2.2)更好的转移率，并且彼此具有可比性。每种转移方法都在神经胶质细胞和神经细胞的不同群体中产生了阳性信号。这些数据为切片培养的最佳转染法提供了基础，使治疗结构的开发成为可能，并支持了使用脑切片培养成功改进体内使用的非病毒输送方法的想法。
Organotypic slice cultures from postnatal day 12 mouse cerebellum were transfected using three nonviral methods: biolistics (gene gun), lipotransfection, and electroporation. The plasmid transferred, pHD17-25Q-GFP, encoded a fusion protein with a green fluorescent protein (GFP) component. Optimal conditions for both lipotransfection and electroporation are the same as those previously found in live animal models. Electroporation (26 +/- 6) and biolistics (34 +/- 4.4) provide a better rate of transfer than lipotransfection (15 +/- 2.2) in slice cultures and are comparable to each other. Each of the transfer methods produced positive signals in a heterogeneous population of glial and neuronal cells. These data provide a base for optimal transfection of slice cultures, allowing the development of therapeutic constructs, and support the idea that successful refinement of nonviral delivery methods for in vivo use is possible using brain slice cultures.