Kalebic Nereo, Langen Barbara, Helppi Jussi, Kawasaki Hiroshi, Huttner Wieland B
Max Planck Institute of Molecular Cell Biology and Genetics; Human Technopole;
Max Planck Institute of Molecular Cell Biology and Genetics; Landesdirektion Sachsen.
J Vis Exp. 2020 May 6(159). doi: 10.3791/61171.
Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.
在分析单个基因在哺乳动物发育过程中的作用时,在胚胎发育期间体内操纵基因表达是首选方法。子宫内电穿孔是在体内操纵胚胎哺乳动物大脑中基因表达的关键技术。本文介绍了一种对小型食肉动物雪貂胚胎新皮层进行子宫内电穿孔的方案。雪貂越来越多地被用作新皮层发育的模型,因为它的新皮层呈现出一系列在人类和非人类灵长类动物中也存在,但在啮齿动物模型(如小鼠或大鼠)中不存在的解剖学、组织学、细胞和分子特征。子宫内电穿孔在胚胎第33天(E33)进行,这是雪貂神经发生中期阶段。子宫内电穿孔的目标是大脑侧脑室内衬的神经祖细胞。在神经发生过程中,这些祖细胞产生所有其他神经细胞类型。这项工作展示了在E37、出生后第1天(P1)和P16的代表性结果和分析,分别对应于子宫内电穿孔后4、9和24天。在早期阶段,靶向细胞的后代主要由各种神经祖细胞亚型组成,而在后期阶段,大多数标记细胞是有丝分裂后的神经元。因此,子宫内电穿孔能够研究基因操纵对各种类型神经细胞的细胞和分子特征的影响。通过其对各种细胞群体的作用,子宫内电穿孔还可用于操纵雪貂新皮层的组织学和解剖学特征。重要的是,所有这些作用都是急性的,并且是在用户确定的时空特异性下进行的。