7] Use of transgenic mice to study activation of retinoic acid-responsive promoters

Abstract

Transgenic systems are invaluable in studies of function of retinoic acids (RAs). The spatial-temporal distribution of RAs within the embryo or a given tissue is difficult to assess because it involves laborious and tissue-disrupting analytical methods such as high-performance liquid chromatography (HPLC). As an alternative, relative RA concentrations can be measured in situ using RA-inducible promoter sequences fused to a reporter gene. Transgenic approaches may be also used to identify novel RA-dependent genes. Gene traps are used to identify novel genes that are important for development. In this strategy, pluripotent mouse embryonic stem (ES) cells are electroporated with a gene trap vector. The vector consists of a promoterless reporter gene (typically β -galactosidase), preceded by a splice-acceptor site. A selection marker provides means to select clones derived from ES cells that have integrated the construct into their genomes. Alternatively, the β -galactosidase and neomycin phosphotransferase activities can be contained in a single fusion protein. The activity of the reporter gene can be visualized by histological staining. Stained clones contain β -galactosidase-tagged genes, which are expressed in the ES cells. A modification of this technique allows the investigator to identify a group of genes that is inducible by a particular soluble factor.