Cytoplasmic injection of murine zygotes with Sleeping Beauty transposon plasmids and minicircles results in the efficient generation of germline transgenic mice
Cytoplasmic injection of murine zygotes with Sleeping Beauty transposon plasmids and minicircles results in the efficient generation of germline transgenic mice
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DOI：
10.1002/biot.201500218
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发表时间：
2016-01-01
影响因子：
4.7
通讯作者：
Kues, Wilfried A.
中科院分区：
文献类型：
--
作者：
Garrels, Wiebke;Talluri, Thirumala R.;Kues, Wilfried A.
小鼠的转基因是了解基因功能和基因组组织的重要工具。在这里，我们描述了一种简化的显微注射方案，用于在使用不同拓扑的二进制睡美人转座子构建的小鼠模型中有效地进行生殖系转基因和持续的转基因表达。该方案是基于将编码睡美人转座酶和转座子结构的超螺旋质粒或微环共同注射到小鼠受精卵的细胞质中。重要的是，这种简化的注射避免了对脆弱的原核膜的机械穿透，导致处理后胚胎的存活率更高，注射速度更快。转座酶翻译后，转座酶催化的转座进入基因组，导致稳定的转基因动物携带单体转基因。综上所述，胞质注射二元转座子构建是一种可行的、基于质粒的、简化的微注射方法来产生转基因小鼠。组件的模块化设计允许不同转座子的多路复用，并在一步内产生多个转座子转基因小鼠。
Transgenesis in the mouse is an essential tool for the understanding of gene function and genome organization. Here, we describe a simplified microinjection protocol for efficient germline transgenesis and sustained transgene expression in the mouse model employing binary Sleeping Beauty transposon constructs of different topology. The protocol is based on co-injection of supercoiled plasmids or minicircles, encoding the Sleeping Beauty transposase and a transposon construct, into the cytoplasm of murine zygotes. Importantly, this simplified injection avoids the mechanical penetration of the vulnerable pronuclear membrane, resulting in higher survival rates of treated embryos and a more rapid pace of injections. Upon translation of the transposase, transposase-catalyzed transposition into the genome results in stable transgenic animals carrying monomeric transgenes. In summary, cytoplasmic injection of binary transposon constructs is a feasible, plasmid-based, and simplified microinjection method to generate genetically modified mice. The modular design of the components allows the multiplexing of different transposons, and the generation of multi-transposon transgenic mice in a single step.