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Vol. 11, Issue 11, 1871-1877, November 2001

METHODS
Genomewide Trapping of Genes that Encode Secreted and Transmembrane Proteins Repressed by Oncogenic Signaling

Mathias Gebauer,1 Harald von Melchner,2,3 and Thomas Beckers1

1 ASTA Medica AG, Department of Cancer Research and 2 Laboratory for Molecular Hematology, University of Frankfurt Medical School, Frankfurt am Main, Germany

A retroviral gene trap containing a human CD2 cell surface antigen/neomycin-phosphotransferase fusion gene in the U3 region of its LTR (U3Ceo) was used to screen the mammalian genome for genes encoding secreted and/or transmembrane proteins that are repressed by oncogenic transformation. From an integration library consisting of cells transformable by the insulin-like growth factor 1 (IGF-1), a collection of neomycin resistant (NeoR) clones was obtained; 86% also expressed the CD2 cell surface antigen. Molecular analysis of a random sample of NeoR clones revealed that the U3Ceo gene trap preferentially disrupted genes coding for secreted and transmembrane proteins. In each case, the signal sequence of the endogenous gene was fused in-frame to the CD2/neomycin-phosphotransferase reporter gene due to a cryptic splice acceptor site embedded in the coding region of the CD2 cDNA. When the library was transformed by IGF-1 and selected against CD2 expression, integrations were obtained in genes that are repressed by transformation. Molecular analysis of six randomly chosen integrations revealed that, in each case, U3Ceo captured a signal sequence from proteins involved in oncogenic transformation and metastatic spread.

3 Corresponding author.

11:1871-1877 ©2001 by Cold Spring Harbor Laboratory Press  ISSN 1088-9051/01 $5.00
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