EFFICIENCY ASSESSMENT OF THE GENE TRAP APPROACH

The trapping of genes in murine embryonic stem (ES) cells offers three features in one experimental approach: 1) analysis of the expression patterns of unknown genes by using a simple staining method, 2) rapid cloning of unknown genes, and 3) generation of mutant mouse lines. We performed a gene trap screen aimed at the discovery of new genes regul ating embryonic development. We have processed 209 gene trap events fo r expression patterns in chimeric murine embryos. Randomly tested, bet a-galactosidase-positive ES cell clones resulted in vivo in 35% gene t rap events showing no beta-galactosidase activity, 39% gene trap event s with ubiquitous beta-galactosidase activity, and 26% gene trap event s showing beta-galactosidase activity restricted to specific cell type s or organs. In vitro preselection reduced gene trap events with ubiqu itous beta-galactosidase activity to 10% and increased the gene trap e vents with restricted beta-galactosidase activity to 64%, making the s creening procedure for genes expressed in a restricted manner 2.5-fold more efficient. In five of the seven gene trap insertions into genes in which the expression pattern during embryogenesis was known, the be ta-galactosidase marker gene reproduced faithfully the expression patt ern of the trapped gene, 5'-Rapid amplification of cDNA ends (5'-RACE) of 28 gene trap events revealed 19 novel mouse genes, 8 known mouse g enes, and 1 random transsplicing event. Twelve of the 25 mouse lines t hat crossed to homozygosity showed overt abnormalities. The genomic st ructure was investigated in four of these gene trap events, which caus ed obvious abnormalities. In all four cases, the splice-acceptor gene trap construct was inserted into an exon, One of the 13 gene trap even ts that did not result in overt abnormalities was examined for the pre sence of wild-type mRNA. Homozygous animals were found to produce norm al levels of wild-type mRNA. Evidently, gene trapping does not always provide all three of the features mentioned above. In this paper, we d iscuss the efficiency of gene trapping and ways in which some problems may be overcome. (C) 1998 Wiley-Liss, Inc.