Johansson Torbjörn, Broll Ilja, Frenz Theresa, Hemmers Saskia, Becher Burkhard, Zeilhofer Hanns Ulrich, Buch Thorsten
Institute of Pharmacology and Toxicology, University of Zürich, Zürich, Switzerland.
Genesis. 2010 Apr;48(4):264-80. doi: 10.1002/dvg.20612.
Transgenic mice are highly valuable tools for biological research as they allow cell type-specific expression of functionally instrumental genes. In this protocol, the generation of bacterial artificial chromosome (BAC) transgenic constructs is described. We give an overview of different transgenic inserts, such as fluorescent proteins (alone or in combination with Cre variants), diphtheria toxin receptor, lacZ, and light-activated ion channels. The most reliable and versatile approach to express these genes is by using BACs, which allow "highjacking" of the expression pattern of a gene without characterizing its transcriptional control elements. Here, we describe the necessary cloning techniques compared with conventional transgenesis. With the provided "toolbox" of already available transgene constructs, the generation of the BAC transgenes is made easy and rapid. We provide a comprehensive outline how to insert the different transgenes into a chosen BAC by either ET cloning or recombineering. We also describe in detail the methods to identify the correct insertion and the integrity of the final BAC construct, and finally, the preparation of the BAC DNA for oocyte injection is described.
转基因小鼠是生物学研究中非常有价值的工具，因为它们能够实现功能相关基因的细胞类型特异性表达。在本方案中，将描述细菌人工染色体（BAC）转基因构建体的生成。我们概述了不同的转基因插入片段，如荧光蛋白（单独或与Cre变体组合）、白喉毒素受体、lacZ和光激活离子通道。表达这些基因最可靠、最通用的方法是使用BAC，它可以在不表征其转录控制元件的情况下“劫持”基因的表达模式。在这里，我们将与传统转基因技术相比，描述必要的克隆技术。利用已有的转基因构建体“工具箱”，BAC转基因的生成变得轻松快捷。我们提供了一个全面的概述，说明如何通过ET克隆或重组工程将不同的转基因插入到选定的BAC中。我们还详细描述了鉴定正确插入和最终BAC构建体完整性的方法，最后，描述了用于卵母细胞注射的BAC DNA的制备方法。
