Building a Zoo of Mice for Genetic Analyses: A Comprehensive Protocol for the Rapid Generation of BAC Transgenic Mice
Building a Zoo of Mice for Genetic Analyses: A Comprehensive Protocol for the Rapid Generation of BAC Transgenic Mice
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DOI：
10.1002/dvg.20612
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发表时间：
2010-04-01
期刊：
影响因子：
1.5
通讯作者：
Buch, Thorsten
中科院分区：
文献类型：
--
作者：
Johansson, Torbjoern;Broll, Ilia;Buch, Thorsten
转基因小鼠是生物学研究非常有价值的工具，因为它们允许功能性工具基因的细胞类型特异性表达。在该协议中，描述了细菌人工染色体（BAG）转基因构建体的生成。我们概述了不同的转基因插入片段，例如荧光蛋白（单独或与 Cre 变体组合）、白喉毒素受体、lacZ 和光激活离子通道。表达这些基因的最可靠和最通用的方法是使用 BAC，它允许“劫持”基因的表达模式，而不表征其转录控制元件。在这里，我们描述了与传统转基因相比必要的克隆技术。利用所提供的已有转基因构建体的“工具箱”，BAG转基因的生成变得容易和快速。我们提供了如何通过 ET 克隆或重组工程将不同转基因插入所选 BAG 的全面概述。我们还详细描述了识别最终 BAG 构建体的正确插入和完整性的方法，最后描述了用于卵母细胞注射的 BAG DNA 的制备。创世纪 48:264-280, 2010。(C) 2010 Wiley-Liss, Inc.
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 (BAG) 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 BAG transgenes is made easy and rapid. We provide a comprehensive outline how to insert the different transgenes into a chosen BAG by either ET cloning or recombineering. We also describe in detail the methods to identify the correct insertion and the integrity of the final BAG construct, and finally, the preparation of the BAG DNA for oocyte injection is described. genesis 48:264-280, 2010. (C) 2010 Wiley-Liss, Inc.