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 vectorbased
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 Ttrapgt/gt mice. In contrast to Ttrap's documented essential role during nodal and Smad3
controlled zebrafish early embryogenesis, Ttrapgt/gt mice were born with a normal Mendelian
distribution. However, subsequent analysis of these Ttrapgt/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.

Publication year:2009

Keywords:genetrap, TTRAP, mouse genetics