UNEXPECTED BEHAVIOR OF A GENE TRAP VECTOR COMPRISING A FUSION BETWEENTHE SH BLE AND THE LACZ GENES

A new gene trap vector has been designed, comprised of a fusion betwee n the Sh ble gene, which confers resistance to the antibiotic phleomyc in, and the lacZ gene (phleal fusion gene). A synthetic splice accepto r, made of the yeast branchpoint followed by a pyrimidin-rich sequence of 27 nucleotides, is included at the 5' extremity. The linearized ge ne trap vector was introduced into mouse embryonic stem cells (ES cell s), and 40 phleomycin resistant (phleo(r)) cell lines possessing a sin gle copy of the insert were selected. They were stable in expressing t he lacZ gene. Reporter gene expression was studied at days 8.5 and 10. 5 of embryonic development in chimeric embryos obtained after injectio n of phleo(r) ES clones into 8-cell stage embryos. Out of 20 phleal li nes examined, 14 exhibited beta-galactosidase expression at day 10.5. Use of the phleal fusion gene trap vector to select genes expressed in ES cells, therefore, is compatible with the isolation of genes expres sed at midgestation. However, and most intriguingly, 10 out of these 1 4 cell lines (71%) displayed reporter gene expression mostly in heart and liver. Two of them exhibited, in addition, expression in central n ervous system (CNS) or in CNS and limb buds, respectively. Germline ch imeras were subsequently obtained and 15 mouse lines have been establi shed. Intercrosses of animals heterozygous for the insertion revealed a mutant phenotype in several lines. (C) 1996 Wiley-Liss, Inc.