Engineering BAC Reporter Gene Constructs for Mouse Transgenesis

Abstract

A culmination of large-scale ideas and efforts has truly allowed for the use of large genomic DNA clones housed in Bacterial Artificial Chromosome (BAC) vectors for biological research. Fundamental advances that have allowed this to happen include (1) the completion of genome sequencing projects and the establishment of highly annotated web-accessible databases allowing for the rapid identity and purchase of BAC clones containing genes of interest. (2) The generation of methodologies to modify BACs genetically, allowing for the rapid creation of gene targeting constructs or transgenic reporter gene constructs using homologous recombination in bacteria.Recent efforts on our part have capitalized on these advances by using BACs and bacterial recombination methods to generate fluorescent protein reporter transgenic mice to study skeletal biology. The rationale for using BAC genomic DNA clones to engineer reporter gene constructs is based on their much larger size, thus increasing the likelihood that most, if not all, of a gene's respective cis regulator elements are present, giving a truer representation of the endogenous gene's expression. In a relatively short amount of time, we have become extremely proficient at generating BAC reporters. Contrary to the widely perceived notion that working with BACs is complex and difficult, we decided to write this chapter to encourage laboratories that are currently using traditional molecular cloning methods to engineer transgenic DNA constructs to strongly consider learning BAC methodologies. As an example, we walk through the steps we took to generate the transgenic reporter mouse line, Tenascin C (TNC)-mCherry.