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 regulating embryonic development. We have processed 209 gene trap events for expression patterns in chimeric murine embryos. Randomly tested, beta-galactosidase-positive ES cell clones resulted in vivo in 35% gene trap events showing no beta-galactosidase activity, 39% gene trap events with ubiquitous beta-galactosidase activity, and 26% gene trap events showing beta-galactosidase activity restricted to specific cell types or organs. In vitro preselection reduced gene trap events with ubiquitous beta-galactosidase activity to 10% and increased the gene trap events with restricted beta-galactosidase activity to 64%, making the screening 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 beta-galactosidase marker gene reproduced faithfully the expression pattern 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 genes, and 1 random transsplicing event. Twelve of the 25 mouse lines that crossed to homozygosity showed overt abnormalities. The genomic structure was investigated in four of these gene trap events, which caused obvious abnormalities. In all four cases, the splice-acceptor gene trap construct was inserted into an exon. One of the 13 gene trap events that did not result in overt abnormalities was examined for the presence of wild-type mRNA. Homozygous animals were found to produce normal levels of wild-type mRNA. Evidently, gene trapping does not always provide all three of the features mentioned above. In this paper, we discuss the efficiency of gene trapping and ways in which some problems may be overcome.
Voss
A K
AK
Department of Molecular Cell Biology, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany. avoss@gwdg.de
Thomas
T
T
Gruss
P
P
D000818:Animals; D001483:Base Sequence; D002678:Chimera / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant; D005314:Embryonic and Fetal Development / Q000235:genetics; D005260:Female; D015870:Gene Expression; D005821:Genetic Techniques; D006720:Homozygote; D066298:In Vitro Techniques; D051379:Mice; D008817:Mice, Mutant Strains; D016254:Mutagenesis, Insertional; D009154:Mutation; D011247:Pregnancy; D019105:Random Amplified Polymorphic DNA Technique; D013234:Stem Cells; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D004274:DNA, Recombinant; D001616:beta-Galactosidase