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In vivo functional proteomics: mammalian genome annotation using CD-tagging.

J W Jarvik1, G W Fisher, C Shi

  • 1Carnegie Mellon University, Pittsburgh, PA, USA.

Biotechniques
|October 26, 2002
PubMed
Summary
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Researchers tagged genes and proteins in mouse cells using a novel retroviral vector. This method systematically annotates mammalian genomes and proteomes, revealing protein abundance and location in living cells.

Area of Science:

  • Molecular Biology
  • Genomics
  • Proteomics

Background:

  • Understanding gene and protein function is crucial for deciphering cellular processes.
  • Existing methods for functional annotation can be limited in scope or application.
  • Developing systematic approaches for genome-wide analysis in living cells is essential.

Purpose of the Study:

  • To develop and validate a novel retroviral vector for tagging genes and proteins.
  • To systematically analyze the functional annotation of mammalian genomes and proteomes.
  • To assess the accuracy of tagged protein abundance and cellular localization.

Main Methods:

  • Utilized a self-inactivating CD-tagging retroviral vector for gene and protein tagging.
  • Introduced epitope and Green Fluorescent Protein (GFP) tags into NIH 3T3 cells.

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  • Isolated hundreds of cell clones with distinct GFP fluorescence patterns.
  • Performed molecular analysis to identify tagged genes and proteins.

    • Main Results:

    • Successfully tagged a diverse range of known and newly discovered genes and proteins.
    • Observed distinct GFP fluorescence patterns across isolated cell clones.
    • Demonstrated that tagged proteins accurately reflect the abundance and cellular location of their untagged counterparts in most cases.

    Conclusions:

    • The developed CD-tagging retroviral vector enables systematic functional annotation of mammalian genomes and proteomes.
    • This technique allows for the study of gene and protein function in living cells.
    • The method provides a reliable way to assess protein abundance and localization, aiding in the understanding of cellular mechanisms.