High-throughput trapping of secretory pathway genes in mouse embryonic stem cells.
High-throughput gene trapping is a random approach for inducing insertional mutations across the mouse genome. This approach uses gene trap vectors that simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag for the rapid identification of the disrupted gene. Gene trapping has been used by both public and private institutions to produce libraries of embryonic stem (ES) cells harboring mutations in single genes. Presently, approximately 66% of the protein coding genes in the mouse genome have been disrupted by gene trap insertions. Among these, however, genes encoding signal peptides or transmembrane domains (secretory genes) are underrepresented because they are not susceptible to conventional trapping methods. Here, we describe a high-throughput gene trapping strategy that effectively targets secretory genes. We used this strategy to assemble a library of ES cells harboring mutations in 716 unique secretory genes, of which 61% were not trapped by conventional trapping, indicating that the two strategies are complementary. The trapped ES cell lines, which can be ordered from the International Gene Trap Consortium (http://www.genetrap.org), are freely available to the scientific community.
Department of Molecular Hematology, University of Frankfurt Medical School, Frankfurt am Main, Germany.
Silke
S
De-Zolt
Frank
F
Schnütgen
Claudia
C
Seisenberger
Jens
J
Hansen
Melanie
M
Hollatz
Thomas
T
Floss
Patricia
P
Ruiz
Wolfgang
W
Wurst
Harald
H
von Melchner
D000818:Animals; D018801:CD2 Antigens / Q000378:metabolism; D002460:Cell Line; D004622:Embryo, Mammalian / Q000166:cytology*; D008565:Membrane Proteins / Q000737:chemistry; D051379:Mice / Q000196:embryology / Q000235:genetics*; D008822:Mice, Transgenic; D016254:Mutagenesis, Insertional / Q000379:methods*; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D013234:Stem Cells / Q000378:metabolism*
D018801:CD2 Antigens; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011506:Proteins