Welch Elaine L, Eno Celeste C, Nair Sreelaja, Lindeman Robin E, Pelegri Francisco
Laboratory of Genetics, University of Wisconsin-Madison.
Department of Biological Sciences, Tata Institute of Fundamental Research.
J Vis Exp. 2017 Apr 22(122):55213. doi: 10.3791/55213.
Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.
动物胚胎发育最早阶段发生的细胞事件是由沉积在发育中的卵母细胞中的母源基因产物驱动的。由于这些事件依赖于通常在受精后很快就起作用的母源产物——这些产物预先存在于卵子内部，因此涉及将试剂注入受精卵的标准表达和功能降低方法通常无效。相反，这种操作必须在卵子发生过程中，在母源产物积累之前或期间进行。本文详细描述了未成熟斑马鱼卵母细胞的体外成熟及其随后的体外受精方案，产生能存活至成年的有活力胚胎。这种方法允许在卵子发生过程中对母源产物进行功能操作，例如用于表型拯救的产物表达和标记构建体可视化，以及通过反向遗传学试剂降低基因功能。
