Chekuru Avinash, Kuscha Veronika, Hans Stefan, Brand Michael
Technische Universität Dresden, Biotechnology Center and DFG-Center for Regenerative Therapies Dresden Cluster of Excellence, Tatzberg 47-49, 01307, Dresden, Germany.
Methods Mol Biol. 2017;1642:87-97. doi: 10.1007/978-1-4939-7169-5_6.
Cre-mediated site-specific recombination has emerged as an indispensable tool for the precise manipulation of genomes allowing lineage-tracing studies, temporal and spatial misexpressions, and in particular the generation of conditional knockout alleles. Previously, we and others showed that Cre and its ligand-inducible variant CreER are also highly efficient in the developing and adult zebrafish. The number of Cre driver and effector lines is currently still limited in zebrafish. However, the recent advent of novel genome editing tools such as TALEN and CRISPR/Cas will significantly increase interest in the conditional Cre/lox-technology in this organism. The considerations of basic transgene design and subsequent transgenesis have been addressed elsewhere. Here we outline practical experimental steps for transient functionality tests of CreER driver and effector constructs. In addition, we introduce detailed protocols to elicit CreER-mediated recombination in vivo at embryonic as well as adult stages.
Cre介导的位点特异性重组已成为精确操纵基因组不可或缺的工具，可用于谱系追踪研究、时空错误表达，特别是条件性敲除等位基因的产生。此前，我们和其他人表明，Cre及其配体诱导变体CreER在发育中的斑马鱼和成年斑马鱼中也具有很高的效率。目前，斑马鱼中Cre驱动和效应系的数量仍然有限。然而，诸如TALEN和CRISPR/Cas等新型基因组编辑工具的出现，将显著增加人们对这种生物体中条件性Cre/lox技术的兴趣。基本转基因设计和后续转基因的相关考虑在其他地方已有论述。在此，我们概述了CreER驱动和效应构建体瞬时功能测试的实际实验步骤。此外，我们还介绍了在胚胎期和成年期体内引发CreER介导重组的详细方案。
