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Resources for proteomics in mouse embryonic stem cells

Nature Methods volume 8, pages 103–104 (2011)Cite this article

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To the Editor:

IKMC resources currently contain 25,130 tagging-compatible ESC lines representing 3,695 individual genes ( Supplementary Table 1 ). These lines have FlipRosaβgeo insertions in the first intron of genes, either downstream of the first noncoding exons or of exons that encode relatively short peptides without apparent functional domains. To knock a sequence encoding a protein tag into these loci, we designed an exchange cassette consisting of a hygromycin resistance gene (hygromycin phosphotransferase; hygro) fused via a 2A virus polyprotein cleavage sequence (P2A) to a modified N-terminal localization and affinity purification (nLAP) tag encoding enhanced GFP (EGFP)4. This hygro-P2A-nLAP cassette includes splice acceptor (SA) and splice donor (SD) sites upstream and downstream of the cassette, respectively (Fig. 1a); therefore it is a portable exon. For RMCE, the tagging exon was flanked by heterotypic FLP recombinase target sequences identical to those inserted by the gene trap (Fig. 1a). RMCE induces the expression of a fusion transcript in which the tagging exon is spliced to the endogenous exons of the trapped gene. Translation yields a protein that is cleaved at the P2A site, so that the tagged endogenous protein is expressed independently (Fig. 1a).

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Figure 1: Proteome analysis in trapped ESC lines.

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Acknowledgements

We thank L. von Melchner for useful comments and for help in editing the manuscript, and J. Mühl, A.-T. Tieu, R. Eitz, D. Herold and H. Grunert for technical assistance. This work was supported by grants from the Bundesministerium für Bildung und Forschung to the NGFNplus-DiGtoP consortium (01GS0858), the Deutsche Forschungsgemeinschaft to H.v.M. (ME 82/5-1) and the European Union (EUCOMM project LSHG-CT-2005-018931 to H.v.M. and W.W.).

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Authors and Affiliations

  1. Department for Molecular Hematology, University of Frankfurt Medical School, Frankfurt am Main, Germany

    Frank Schnütgen, Franziska Ehrmann & Harald von Melchner

  2. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

    Ina Poser, Ingrid deVries & Anthony Hyman

  3. Max Planck Institute of Biochemistry, Martinsried, Germany

    Nina C Hubner & Matthias Mann

  4. Institute for Developmental Genetics Helmholtz Zentrum München, Germany

    Jens Hansen, Thomas Floss & Wolfgang Wurst

  5. German Center for Neurodegenerative Diseases, Technische Universität München, Neuherberg, Germany

    Wolfgang Wurst

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  1. Frank Schnütgen
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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 2–3, Supplementary Methods (PDF 3163 kb)

Supplementary Table 1

List of tagging compatible gene trap lines. (XLS 974 kb)

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Schnütgen, F., Ehrmann, F., Poser, I. et al. Resources for proteomics in mouse embryonic stem cells. Nat Methods 8, 103–104 (2011). https://doi.org/10.1038/nmeth0211-103

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