Haddad-Tóvolli Roberta, Szabó Nora-Emöke, Zhou Xunlei, Alvarez-Bolado Gonzalo
Institute of Anatomy and Cell Biology, University of Heidelberg.
J Vis Exp. 2013 Jul 24(77):50412. doi: 10.3791/50412.
Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.
对发育中的哺乳动物大脑特定区域进行基因改造是一种非常强大的实验方法。然而，生成新的小鼠突变体往往极其缓慢。已证明在子宫内发育的小鼠大脑中进行操作并使其在术后具有合理的存活率是可行的。尽管如此，关于这一程序的结果几乎仅报道于发育中大脑最表层且最易触及的部分，即皮层。丘脑是一个更狭窄且更靠内侧的区域，已证明更难靶向。转染到更深的核团，尤其是下丘脑的核团，可能是最具挑战性的，因此报道的结果非常少。在此，我们展示一种通过电穿孔靶向整个下丘脑神经上皮或其部分区域（下丘脑区域）进行转染的程序。我们方法的关键在于更长的麻醉时间、在第三脑室注射以及电极的合适种类和定位。此外，我们展示了对最隐蔽的下丘脑核团乳头体进行靶向及后续组织学分析的结果。
