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Establishment of a gene-trap sequence tag library to generate mutant mice from embryonic stem cells

Nature Genetics volume 24, pages 13–14 (2000)Cite this article

Nucleotide sequences of most vertebrate genes will be available in the next few years as ESTs and genomic sequences. This information will allow the deduction of encoded proteins, possibly providing clues to their functions in vivo. To determine unequivocally the physiological roles of all genes and in particular those of medical importance, mutational analyses must be performed. Large-scale random mutagenesis screens, which allow the systematic mutation of all genes, are a valuable approach to this task1,2,3,4,5,6,7,8,9. To complement the sequencing efforts, we established a consortium generating a reference library of gene-trap sequence tags (GTST) which eventually will represent insertional mutations for most mammalian genes expressed in mouse embryonic stem (ES) cells.

We generated approximately 12,000 targeted ES cell lines using both vector types. We performed nucleotide sequencing using a semi-automated 5´-RACE–PCR protocol. Of 3,242 clones sequenced so far, 1,840 yielded informative sequences. For the remaining clones the PCR products were either too short or unusual splicing events occurred, resulting in non-useful clones which were discarded. Comparison with NCBI GenBank data using BlastN or BlastX homology programs showed similarity to non-redundant gene sequences (e≤10−10) in 868 cases (47%) and homology to human or mouse ESTs (e≤10−10) in 439 cases (24%). We obtained novel gene sequences for 533 clones (29%). These data indicate that most trapped genes have previously been identified, but in most instances their functions have not been assessed. We found 521 and 692 clones with matches to sequences of human and mouse UniGene clusters, respectively. We mapped 257 identified gene sequences to human chromosomes by relating the human UniGene clusters to the Genemap 98 radiation-hybrid mapping data. On the basis of these results, GT integrations appear to be randomly distributed on different chromosomes.

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Figure 1: Presentation of gene trap vectors and integration sites.

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Acknowledgements

We thank A. von Holst for critically reading the manuscript; B. Skarnes and E. Ruley for pGT1.8 and U3βgeo gene trap vectors; S. Hennig and A. Hewelt for computer support; T. Gellert, A. Gatlik, S. Bourier, C. Werner, B. Schroeter, N. Lehmann, N. Daigle, T. Engist and B. Fella for expert technical assistance; and S. Rengsberger and A. Maier for secretarial work and artwork. This consortium has been supported by the Bundesministerium für Bildung und Forschung within the framework of the German Human Genome Project.

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Authors and Affiliations

  1. Max-Planck-Institute for Molecular Genetics, Berlin, Germany

    Michael V. Wiles, Patricia Ruiz & Hans Lehrach

  2. GSF-National Research Center for Environment and Health, Institute of Mammalian Genetics, Neuherberg, Germany

    Franz Vauti, Thomas Metz & Wolfgang Wurst

  3. Laboratory for Molecular Hematology, University of Frankfurt Medical School, Frankfurt am Main, Germany

    Jürgen Otte & Harald von Melchner

  4. Department of Developmental Biology, Max-Planck-Institute of Immunobiology, Freiburg, Germany

    Ernst-Martin Füchtbauer & Annette Füchtbauer

  5. Institute of Molecular and Structural Biology, Aarhus University, Aarhus, Denmark

    Ernst-Martin Füchtbauer

  6. Department of Cell and Molecular Biology, Institute of Biochemistry and Biotechnology, TU Braunschweig, Braunschweig, Germany

    Hans-Henning Arnold

  7. Max-Planck-Institute of Psychiatry, Molecular Neurogenetics , Munich, Germany

    Wolfgang Wurst

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  1. Michael V. Wiles
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Correspondence to Wolfgang Wurst.

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Wiles, M., Vauti, F., Otte, J. et al. Establishment of a gene-trap sequence tag library to generate mutant mice from embryonic stem cells. Nat Genet 24, 13–14 (2000). https://doi.org/10.1038/71622

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