In utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells.
In utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells.
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DOI：
10.3791/1333
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发表时间：
2009-09-24
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影响因子：
--
通讯作者：
Mason, Carol A
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--
作者：
Petros, Timothy J;Rebsam, Alexandra;Mason, Carol A
视网膜及其唯一的输出神经元--视网膜神经节细胞(RGC)是研究细胞分化、轴突引导、视网膜组织和突触形成等生物学问题的极佳模型。一个缺点是无法在活体内有效和可靠地操纵视网膜节细胞的基因表达，特别是在原本可以到达的小鼠视觉通路中。转基因小鼠可以用来操纵基因表达，但这种方法通常昂贵、耗时，而且会产生不想要的副作用。在鸡体内，电穿孔被用来操纵RGC中的基因表达，以检测视网膜和RGC的发育。虽然类似的电穿孔技术在体外已经在新生小鼠幼鼠、成年大鼠和胚胎小鼠视网膜中开发出来，但这些策略都不能完全表征RGC的发育和体内轴突的投射。为此，我们开发了两种电穿孔的应用，一种在子宫内，另一种在体外，专门针对胚胎小鼠视网膜神经节细胞。通过宫内视网膜电穿孔，我们可以错误表达或下调RGC中的特定基因，并在体内跟随其轴突投射穿过视觉通路，从而能够检查中间目标(如视交叉)或目标区域(如外侧膝状体)的指导决策。在野生型背景下干扰RGC子集的基因表达有助于理解整个视网膜通路中的基因功能。此外，我们还开发了一种配套技术来分析RGC轴突的体外生长。我们在体外对胚胎头部进行电穿孔，收集并孵化整个视网膜，然后在几天后准备这些视网膜的外植体。视网膜外植体可用于各种体外实验，以检测电穿孔RGC轴突对引导线索或其他因素的反应。总而言之，这套技术增强了我们在RGC中错误表达或下调基因的能力，应该会极大地帮助研究RGC的发育和轴突投射。
The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups, adult rats, and embryonic murine retinae in vitro, none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs. With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.