Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon. BMC Biotechnol 2006 Jun 26;6:30
Date
06/28/2006Pubmed ID
16800892Pubmed Central ID
PMC1557845DOI
10.1186/1472-6750-6-30Scopus ID
2-s2.0-33748186110 (requires institutional sign-in at Scopus site) 36 CitationsAbstract
BACKGROUND: Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB) transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern.
RESULTS: Here we report the systematic development and testing of a transposon-based gene-trap system incorporating the doxycycline-repressible Tet-Off (tTA) system that is capable of activating the expression of genes under control of a Tet response element (TRE) promoter. We demonstrate that the gene trap system is fully functional in vitro by introducing the "gene-trap tTA" vector into human cells by transposition and identifying clones that activate expression of a TRE-luciferase transgene in a doxycycline-dependent manner. In transgenic mice, we mobilize gene-trap tTA vectors, discover parameters that can affect germline mobilization rates, and identify candidate gene insertions to demonstrate the in vivo functionality of the vector system. We further demonstrate that the gene-trap can act as a reporter of endogenous gene expression and it can be coupled with bioluminescent imaging to identify genes with tissue-specific expression patterns.
CONCLUSION: Akin to the GAL4/UAS system used in the fly, we have made progress developing a tool for mutating and revealing the expression of mouse genes by generating the tTA transactivator in the presence of a secondary TRE-regulated reporter molecule. A vector like the gene-trap tTA could provide a means for both annotating mouse genes and creating a resource of mice that express a regulable transcription factor in temporally- and tissue-specific patterns for conditional gene expression studies. These mice would be a valuable resource to the mouse genetics community for purpose of dissecting mammalian gene function.
Author List
Geurts AM, Wilber A, Carlson CM, Lobitz PD, Clark KJ, Hackett PB, McIvor RS, Largaespada DAAuthor
Aron Geurts PhD Professor in the Physiology department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsDNA Transposable Elements
Gene Expression
Gene Transfer Techniques
Genes, Reporter
Genetic Techniques
Mice
Mice, Transgenic
Mutagenesis, Insertional
