Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina.
Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina.
复制标题
DOI：
10.1186/1471-2202-2-2
复制
发表时间：
2001
期刊：
影响因子：
2.4
通讯作者：
Finnegan SG
中科院分区：
文献类型：
--
作者：
Horbinski C;Stachowiak MK;Higgins D;Finnegan SG
已被证明对细胞系成功的化学转染方法通常不适用于神经元的原代培养物。然而，最近的数据表明，阳离子聚乙烯亚胺（PEI）的线性聚合物可以促进神经元对核酸的摄取。因此，我们研究了商业PEI制剂允许将外源基因引入有丝分裂后哺乳动物神经元的能力。从围产期大鼠幼仔获得交感神经元，并在无非神经元细胞的情况下在体外维持5天。然后用不同量的编码E. coli β-半乳糖苷酶或增强型绿色荧光蛋白（EGFP）。在1 μg/ml质粒DNA和5 μg/ml PEI的条件下观察到最佳转染效率。β-半乳糖苷酶的表达既快速又稳定，在6小时内开始并持续至少21天。通过组织化学和抗体染色评估，在72小时内获得最大产量，其中约9%的神经元表达β-半乳糖苷酶。实现了编码报告基因的两个质粒的共转染。来自成人视网膜培养物的有丝分裂后神经元也表现出使用PEI作为载体摄取和表达外源DNA的能力。这些数据表明，PEI是一个有用的代理质粒编码的基因在神经元培养物中的稳定表达。
Chemical methods of transfection that have proven successful with cell lines often do not work with primary cultures of neurons. Recent data, however, suggest that linear polymers of the cation polyethyleneimine (PEI) can facilitate the uptake of nucleic acids by neurons. Consequently, we examined the ability of a commercial PEI preparation to allow the introduction of foreign genes into postmitotic mammalian neurons. Sympathetic neurons were obtained from perinatal rat pups and maintained for 5 days in vitro in the absence of nonneuronal cells. Cultures were then transfected with varying amounts of a plasmid encoding either E. coli β-galactosidase or enhanced green fluorescence protein (EGFP) using PEI. Optimal transfection efficiency was observed with 1 μg/ml of plasmid DNA and 5 μg/ml PEI. Expression of β-galactosidase was both rapid and stable, beginning within 6 hours and lasting for at least 21 days. A maximum yield was obtained within 72 hours with ∼ 9% of the neurons expressing β-galactosidase, as assessed by both histochemistry and antibody staining. Cotransfection of two plasmids encoding reporter genes was achieved. Postmitotic neurons from adult human retinal cultures also demonstrated an ability to take up and express foreign DNA using PEI as a vector. These data suggest that PEI is a useful agent for the stable expression of plasmid-encoded genes in neuronal cultures.
登录
查看更多内容
影响因子：
5.1
作者：
Baker, A;Saltik, M;Cotten, M
通讯作者：
Cotten, M
影响因子：
5.1
作者：
Ogris, M;Brunner, S;Wagner, E
通讯作者：
Wagner, E
影响因子：
4.8
作者：
Bandyopadhyay, P;Ma, XM;Steer, CJ
通讯作者：
Steer, CJ
影响因子：
5.1
作者：
MeunierDurmort, C;Grimal, H;Forest, C
通讯作者：
Forest, C
影响因子：
4.8
作者：
Diebold, SS;Kursa, P;Zenke, M
通讯作者：
Zenke, M