Sanor Lucas D, Flowers G Parker, Crews Craig M
Department of Molecular, Cellular, and Developmental Biology, Yale University.
Department of Molecular, Cellular, and Developmental Biology, Yale University;
J Vis Exp. 2020 Jan 29(155). doi: 10.3791/60156.
A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as axolotls, to regenerate entire limbs as adults. Here, we outline techniques used to generate chimeric axolotls with Cas9-mutagenized haploid forelimbs that can be used for exploring gene function and the fidelity of limb regeneration. We combine several embryological and genetic techniques, including haploid generation via in vitro activation, CRISPR/Cas9 mutagenesis, and tissue grafting into one protocol to produce a unique system for haploid genetic screening in a model organism of regeneration. This strategy reduces the number of animals, space, and time required for the functional analysis of genes in limb regeneration. This also permits the investigation of regeneration-specific functions of genes that may be required for other essential processes, such as organogenesis, tissue morphogenesis, and other essential embryonic processes. The method described here is a unique platform for conducting haploid genetic screening in a vertebrate model system.
越来越多的遗传技术和资源使研究人员能够探究某些蝾螈物种（如美西螈）成年后再生整个肢体能力的分子起源。在这里，我们概述了用于生成具有Cas9诱变单倍体前肢的嵌合美西螈的技术，这些技术可用于探索基因功能和肢体再生的保真度。我们将几种胚胎学和遗传学技术，包括通过体外激活产生单倍体、CRISPR/Cas9诱变以及组织移植，整合到一个方案中，以创建一个用于在再生模型生物中进行单倍体遗传筛选的独特系统。这种策略减少了肢体再生中基因功能分析所需的动物数量、空间和时间。这也允许对可能是其他重要过程（如器官发生、组织形态发生和其他重要胚胎过程）所必需的基因的再生特异性功能进行研究。这里描述的方法是在脊椎动物模型系统中进行单倍体遗传筛选的独特平台。
