A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome

Abstract

A major challenge of the postgenomic era is the functional characterization of every single gene within the mammalian genome. In an effort to address this challenge, we assembled a collection of mutations in mouse embryonic stem (ES) cells, which is the largest publicly accessible collection of such mutations to date. Using four different gene-trap vectors, we generated 5,142 sequences adjacent to the gene-trap integration sites (gene-trap sequence tags; http://genetrap.de) from >11,000 ES cell clones. Although most of the gene-trap vector insertions occurred randomly throughout the genome, we found both vector-independent and vector-specific integration "hot spots." Because >50% of the hot spots were vector-specific, we conclude that the most effective way to saturate the mouse genome with gene-trap insertions is by using a combination of gene-trap vectors. When a random sample of gene-trap integrations was passaged to the germ line, 59% (17 of 29) produced an observable phenotype in transgenic mice, a frequency similar to that achieved by conventional gene targeting. Thus, gene trapping allows a large-scale and cost-effective production of ES cell clones with mutations distributed throughout the genome, a resource likely to accelerate genome annotation and the in vivo modeling of human disease.

Fig. 1.

Correlation between gene-trap insertions and the number of UniGene clusters per chromosome.

Fig. 2.

Number of gene-trap (GT) insertions into annotated hot spots. All genes with two or more insertions were classified as hot spots. Gene lengths were derived from GenBank.

Fig. 3.

Frequency of gene-trap (GT) insertions into unique UniGene clusters. Data points represent the number of novel UniGene clusters accumulating with every 50 insertions. For further explanation see text.