Premium
Building a zoo of mice for genetic analyses: A comprehensive protocol for the rapid generation of BAC transgenic mice
Author(s) -
Johansson Torbjörn,
Broll Ilja,
Frenz Theresa,
Hemmers Saskia,
Becher Burkhard,
Zeilhofer Hanns Ulrich,
Buch Thorsten
Publication year - 2010
Publication title -
genesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.093
H-Index - 110
eISSN - 1526-968X
pISSN - 1526-954X
DOI - 10.1002/dvg.20612
Subject(s) - recombineering , bacterial artificial chromosome , transgenesis , transgene , biology , cloning (programming) , computational biology , gene , insert (composites) , minigene , genetics , multiple cloning site , genetically modified organism , homologous recombination , genome , expression vector , recombinant dna , computer science , reproductive technology , mechanical engineering , rna , rna splicing , engineering , embryogenesis , programming language
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. genesis 48:264–280, 2010. © 2010 Wiley‐Liss, Inc.