Bland Katherine M, Casey Zachary O, Handwerk Christopher J, Holley Z Logan, Vidal George S
Department of Biology, James Madison University.
Department of Biology, James Madison University;
J Vis Exp. 2017 Nov 17(129):56675. doi: 10.3791/56675.
Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.
通过在少量且稀疏的神经元群体中使基因功能丧失或增强，可以揭示基因的细胞自主神经元功能。要做到这一点，需要构建一种嵌合体，其中基因功能丧失或增强的神经元被基因未受干扰的组织所包围。在这里，我们将Cre-lox重组系统与子宫内电穿孔相结合，以生成可用于研究神经元中基因细胞自主功能的嵌合脑组织。使用子宫内电穿孔将编码荧光标记和Cre重组酶的DNA构建体（可从储存库获得）引入小鼠胚胎大脑中含有侧翼带有loxP位点基因的发育中的皮质神经元。此外，我们描述了对子宫内电穿孔方法的各种改进，这些改进提高了存活率和可重复性。该方法还涉及确定在稀疏或密集神经元群体中Cre介导的重组的效价。标记脑组织的组织学制备不需要（但可以适应）免疫组织化学。所使用的构建体确保荧光标记的神经元携带Cre重组酶基因。组织学制备允许通过对树突和轴突分支以及树突棘进行共聚焦成像来对神经元进行形态分析。由于在稀疏嵌合组织中实现了功能丧失或增强，该方法允许在体内研究基因产物的细胞自主必要性和充分性。
