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Unexpected behavior of a gene trap vector comprising a fusion between the Sh ble and the lac Z genes
Author(s) -
Camus A.,
Kress C.,
Babinet C.,
Barra J.
Publication year - 1996
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/(sici)1098-2795(199611)45:3<255::aid-mrd1>3.0.co;2-r
Subject(s) - biology , gene , fusion gene , reporter gene , microbiology and biotechnology , embryonic stem cell , gene expression , lac operon , mutant , genetics
A new gene trap vector has been designed, comprised of a fusion between the Sh ble gene, which confers resistance to the antibiotic phleomycin, and the lac Z gene (phleal fusion gene). A synthetic splice acceptor, made of the yeast branchpoint followed by a pyrimidin‐rich sequence of 27 nucleotides, is included at the 5′ extremity. The linearized gene trap vector was introduced into mouse embryonic stem cells (ES cells), and 40 phleomycin resistant (phleAL) cell lines possessing a single copy of the insert were selected. They were stable in expressing the lac Z gene. Reporter gene expression was studied at days 8.5 and 10.5 of embryonic development in chimeric embryos obtained after injection of phleoL ES clones into 8‐cell stage embryos. Out of 20 phleal lines examined, 14 exhibited β‐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 expressed at midgestation. However, and most intriguingly, 10 out of these 14 cell lines (71%) displayed reporter gene expression mostly in heart and liver. Two of them exhibited, in addition, expression in central nervous system (CNS) or in CNS and limb buds, respectively. Germline chimeras were subsequently obtained and 15 mouse lines have been established. Intercrosses of animals heterozygous for the insertion revealed a mutant phenotype in several lines. © 1996 Wiley‐Liss, Inc.