Functional Analysis of the Mammalian Genome by Large Scale Gene Trap Mutagenesis

P. Van Sloun
Laboratory for Molecular Hematology, University of Frankfurt Medical School
Frankfurt am Main, Germany.
Telephone: 49-69-6301-6390; Fax: 49-69-6301-7877
Email: vansloun@em.uni-frankfurt.de

Gene trap mutagenesis in mouse embryonic stem (ES) cells is a complementary approach to the functional annotation of the mouse genome. In concert with chemical (ENU) mutagenesis, the approach enables the analysis of gene function in the context of an entire organism and thus furthers our understanding of human disease. Identified disease genes will then greatly assist the process of drug discovery.

We have established a Gene Trap Consortium in the context of the German Human Genome Project (HUGO) with the goal to saturate the mouse genome with insertional mutations. By disrupting genes repeatedly, we anticipate to create an allelic series for each gene expressed in the ES cells.

Within the last four years, the GGTC has generated 14 000 mutant ES cell lines and has identified the gene trap integration sites in 6829 clones. Of the generated gene trap sequence tags (GTSTs), 5269 high quality sequences were used in NCBI-GenBank database searches. The sequences produced 1099 (21%) matches with non-redundant genes, 1552 (29%) matches with expressed sequence tags (EST's), and 2618 (50%) no matches (e£1.0e-30) in part reflecting novel genes. Of all integrations into previously characterized genes, more than 40 occurred in genes involved in human disease. Forty-six ES cell lines were passaged through the germ line and the phenotypes induced by transgenes are being presently assessed.

Data obtained from individual clones, such as GTSTs, blast results and OMIM references are deposited in the GGTC's database which is publicly accessible via http://genetrap.gsf.de. Clones of interest can be identified using blast or keyword search and corresponding cell lines, stored frozen at the GSF in Munich, are freely available upon request.

Since none of the gene trap vectors currently in use incorporates all features one would expect from an ideal vector, the Consortium is actively pursuing the development of novel gene trap vectors. One of these is the U3Ceo vector, a retroviral gene trap designed to specifically trap secreted and transmembrane proteins. Compared to previously reported secretion traps, U3Ceo is at least 4 times as efficient and thus ideally suited for large scale mutagenesis of signal sequence encoding genes.