title: Essential validation of gene trap mouse ES cell lines: a test case with the gene Ttrap.
abstract: Gene trapping in mouse embryonic stem (ES) cells enables near-saturation vector-based insertional mutagenesis across the genome of this model organism. About 135,000 trapped ES cell lines are made available to the scientific community by the International Gene Trap Consortium (IGTC; www.genetrap.org). A search of one of its databases identified an ES cell line (RRS512) with a betaGeo-based gene trap (gt) vector insertion in intron 5 of Ttrap, a gene that encodes an intracellular signalling protein, which is implicated in gastrulation movement and left-right asymmetry in zebrafish embryos. We have determined the exact gt insertion point in the mutant ES cell clone RRS512 and confirmed the production of a chimaeric transcript consisting of the upstream Ttrap exons and the gene trap vector encoded marker/selection fusion sequences. This ES cell line was used to generate heterozygous Ttrap mutant mice, which were further crossed to obtain Ttrap(gt/gt) mice. In contrast to Ttraps documented essential role during nodal and Smad3 controlled zebrafish early embryogenesis, Ttrap(gt/gt) mice were born with a normal Mendelian distribution. However, subsequent analysis of these Ttrap(gt/gt) mice has revealed a duplication of the wild-type Ttrap allele that was already present in the RRS512 cell line. Based on our detailed analysis presented here, we suggest an extensive procedure for the characterization of gene trap ES cell lines prior to generating gene trap mice with these.
authors: Centre of Human Genetics, KULeuven, Belgium.
authors: Liesbeth
authors: L
authors: Vermeire
authors: Abdelilah
authors: A
authors: Ibrahimi
authors: Thierry
authors: T
authors: Voet
authors: Lieve
authors: L
authors: Umans
authors: Kathleen
authors: K
authors: Coddens
authors: Annick
authors: A
authors: Francis
authors: Tom
authors: T
authors: Van de Putte
authors: Leo A
authors: LA
authors: van Grunsven
authors: An
authors: A
authors: Zwijsen
authors: Danny
authors: D
authors: Huylebroeck
authors: Luc
authors: L
authors: Nelles
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005260:Female; D005821:Genetic Techniques; D005822:Genetic Vectors; D017403:In Situ Hybridization; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D011247:Pregnancy; D020133:Reverse Transcriptase Polymerase Chain Reaction; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins / Q000235:genetics*
chemical_list: D017931:DNA Primers; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins