Simultaneous expression of different transgenes in neurons and glia by combining in utero electroporation with the Tol2 transposon-mediated gene transfer system.
Simultaneous expression of different transgenes in neurons and glia by combining in utero electroporation with the Tol2 transposon-mediated gene transfer system.
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DOI：
10.1111/j.1365-2443.2010.01397.x
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发表时间：
2010-05
期刊：
影响因子：
--
通讯作者：
Miura M
中科院分区：
文献类型：
--
作者：
Yoshida A;Yamaguchi Y;Nonomura K;Kawakami K;Takahashi Y;Miura M
在胚胎发生过程中，通过将质粒DNA导入神经祖细胞，在子宫内电穿孔被广泛用于研究神经元的发育和功能。这是一种将质粒DNA导入神经前体的有效而便捷的方法，适用于操纵中枢神经系统细胞中的基因表达。然而，该技术的适用性相对限于神经元研究，因为在胚胎发生期间引入神经祖细胞的质粒DNA被细胞增殖稀释，并且在出生前后产生的胶质细胞中不稳定维持。为了克服这一限制，我们将Tol2转座子系统应用于子宫内电穿孔，该系统将转基因整合到宿主细胞的基因组中。通过该系统，我们证实了转基因在胚胎神经前体、星形胶质细胞和少突胶质细胞的后代中有效维持。利用胶质启动子GFAP和S100β，获得了转基因在胶质细胞中的定向稳定表达，使不同转基因在神经元和胶质细胞中同时表达。在没有神经元迁移缺陷的情况下，实现了胶质细胞靶向表达引起神经元迁移缺陷的转基因。因此，使用Tol2转座子系统结合子宫内电穿孔是研究体内胶质-神经元相互作用的有力方法。
In utero electroporation is widely used to study neuronal development and function by introducing plasmid DNA into neural progenitors during embryogenesis. This is an effective and convenient method of introducing plasmid DNA into neural precursors and is suitable for manipulating gene expression in cells of the CNS. However, the applicability of this technique is comparatively limited to neuronal research, as the plasmid DNA introduced into neural progenitors during embryogenesis is diluted by cell proliferation and is not stably maintained in glial cells generated around and after birth. To overcome this limitation, we applied the Tol2 transposon system, which integrates a transgene into the genome of the host cell, to in utero electroporation. With this system, we confirmed that the transgene was effectively maintained in the progeny of embryonic neural precursors, astrocytes and oligodendrocytes. Using the glial promoters GFAP and S100β, targeted and stable expressions of transgenes in glia were obtained, which enabled the expression of different transgenes simultaneously in neurons and glia. Glia-targeted expression of the transgene that causes neuronal migration defect was achieved without the defect. Thus, use of the Tol2 transposon system in combination with in utero electroporation is a powerful method for studying glia-neuron interactions in vivo.
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DOI：
10.1073/pnas.97.21.11403
发表时间：
2000-10-10
影响因子：
11.1
作者：
Kawakami, K;Shima, A;Kawakami, N
通讯作者：
Kawakami, N
DOI：
10.1006/bbrc.1996.5882
发表时间：
1997-01-13
影响因子：
3.1
作者：
Muramatsu, T;Mizutani, Y;Okumura, J
通讯作者：
Okumura, J
影响因子：
2.7
作者：
Sato, Yuki;Kasai, Toshiharu;Takahashi, Yoshiko
通讯作者：
Takahashi, Yoshiko
影响因子：
1.5
作者：
Hamlet, Michelle R. Johnson;Yergeau, Donald A.;Mead, Paul E.
通讯作者：
Mead, Paul E.
影响因子：
6.2
作者：
Burns, Kevin A.;Murphy, Brian;Kuan, Chia-Yi
通讯作者：
Kuan, Chia-Yi