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

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.
• 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.