Selective disruption of genes expressed in totipotent embryonal stem cells.

Abstract

Two retrovirus promoter trap vectors (U3His and U3Neo) have been used to disrupt genes expressed in totipotent murine embryonal stem (ES) cells. Selection in L-histidinol or G418 produced clones in which the coding sequences for histidinol-dehydrogenase or neomycin-phosphotransferase were fused to sequences in or near the 5' exons of expressed genes, including one in the developmentally regulated REX-1 gene. Five of seven histidinol-resistant clones and three of three G418-resistant clones generated germ-line chimeras. A total of four disrupted genes have been passed to the germ line, of which two resulted in embryonic lethalities when bred to homozygosity. The ability to screen large numbers of recombinant ES cell clones for significant mutations, both in vitro and in vivo, circumvents genetic limitations imposed by the size and long generation time of mice and will facilitate a functional analysis of the mouse genome.

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

“…In pT1␤geo, pT1ATG␤geo, and ROSA␤geo, ␤geo is flanked by an upstream 3Ј splice consensus sequence (splice acceptor) and a downstream polyadenylation site to ensure its activation from integrations into introns (''intron trap'') (11-13). U3␤geo lacks a splice acceptor sequence and therefore is activated mostly from integrations into exons (''exon trap'') (10,14). Because all these gene-trap vectors require a cellular promoter for activation, the maximum number of genomic targets equals the number of expressed genes.…”

“…The E14.1 (129͞Ola), CJ7 (129͞Sv), R1 (129͞Sv ϫ 129͞Sv-CP), and TBV-2 (129͞SvP) ES cell lines were grown on irradiated (x-rays, 32 Gy) or mitomycin C (Sigma)-treated (10 g͞ml for 2.5 h) mouse embryonic fibroblast feeder layers in DMEM (GIBCO͞BRL) supplemented with 10-20% (vol͞vol) preselected and heat-inactivated FCS (Invitrogen), 2 mM glutamine, 1ϫ nonessential amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol (all from Invitrogen), 1,000 units͞ml leukemia inhibitory factor (Esgro, Chemicon), and optionally 5 g͞ml penicillin and streptomycin (GIBCO͞BRL). ES cells were either electroporated with pT1␤geo and pT1ATG␤geo plasmid vectors or infected with U3␤geo and ROSA␤geo retroviruses as described (3,10). Gene-trap-expressing ES cell clones were selected in 200 g͞ml G418 (GIBCO͞BRL), manually picked, expanded, and stored frozen in liquid nitrogen.…”

Identification and validation of PDGF transcriptional targets by microarray-coupled gene-trap mutagenesis

“…To systemically explore PDGF targets and their in vivo functions, we carried out gene-trap mutagenesis, in which a promoterless reporter is introduced into ES cells to randomly tag and mutate genes, integrating expression monitoring, molecular cloning and functional analysis [9][10][11] . Transcriptionally responsive genes can be identified by induction trapping based on the altered expression of the gene-trap reporter on stimulation [12][13][14][15] .…”

Engineering mutations: Deconstructing the mouse gene by gene

“…Gene traps are another tool for producing tagged random mutations in the mouse genome, but unlike gene targeting, they require no previous knowledge of the gene structure (Friedrich and Soriano, 1991;von Melchner et al, 1992;Wurst et al, 1995;Hicks et al, 1997;Hansen et al, 2003;Zambrowicz et al, 2003). Although this method is not generally designed to disrupt a specific target gene, it can be used to generate a library of mutated cell lines.…”

“…Although this method is not generally designed to disrupt a specific target gene, it can be used to generate a library of mutated cell lines. Gene traps were originally developed as an efficient technique to generate mutations in ES cells (Gossler et al, 1989;Friedrich and Soriano, 1991;von Melchner et al, 1992). Gene traps are retroviral-, plasmid-, or transposon-based vectors that disrupt the transcription of a gene upon insertion (for review see .…”