Efficiency assessment of the gene trap approach

Abstract

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, β‐galactosidase‐positive ES cell clones resulted in vivo in 35% gene trap events showing no β‐galactosidase activity, 39% gene trap events with ubiquitous β‐galactosidase activity, and 26% gene trap events showing β‐galactosidase activity restricted to specific cell types or organs. In vitro preselection reduced gene trap events with ubiquitous β‐galactosidase activity to 10% and increased the gene trap events with restricted β‐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 β‐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 trans‐splicing 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. Dev. Dyn. 1998;212:171–180. © 1998 Wiley‐Liss, Inc.

Mena/VASP and αII-Spectrin complexes regulate cytoplasmic actin networks in cardiomyocytes and protect from conduction abnormalities and dilated cardiomyopathy

“…When we analyzed Mena protein levels in our Mena GT/GT mice, we found that specifically in the brain, but not in other organs, the gene trap was “leaky” [27,28], resulting in a low amount of endogenous full length protein (data not shown but compare Mena protein levels in the Mena GT/GT VASP −/− organs, Figure 3A, dKO lanes). We speculated that the residual Mena expression in the brain of Mena GT/GT mice may overcome the embryonic lethal phenotype of Mena/VASP double-deficient mice and crossed the Mena GT/GT animals with our VASP −/− mice [16].…”

MOZ (MYST3, KAT6A) inhibits senescence via the INK4A-ARF pathway

“…A C-terminal tag containing the V5 epitope 66 was introduced into the Moz locus by homologous recombination in ES cells followed by germline chimera production, as described previously. 67 Mice containing the tagged Moz allele were bred to homozygosity to show that the presence of the tag did not impede MOZ function. Although Moz −/− null mice die at or before birth, Moz V5/V5 mice were viable and fertile.…”

A novel triple fusion reporter system for use in gene trap mutagenesis

“…The configuration of bgeo used in our reporters has been extensively reported in gene trap screens, generating very similar percentages of bgal negative clones (40-70% depending on the selection conditions; Bonaldo et al, 1998;Skarnes et al, 1995;Tate et al, 1998;Tzouanacou et al, 2003;Voss et al, 1998;Wilson et al, 1995). This is consistent with our findings, suggesting that there is no adverse effect of the GFP fusion upon bgeo.…”