CD tagging is a genetic technology that provides a novel opportunity to annotate protein function in living cells and organisms. CD tagging is performed by inserting a specially designed DNA sequence (the CD-cassette) into genomic DNA. When the CD-cassette is inserted in the proper orientation in an intron of a transcriptionally active gene, the cassette provides splicing signals that direct the inclusion of a new exon (the guest exon) into the transcript. Translation of the tagged transcript results in the incorporation of a unique peptide tag (the guest peptide) into the protein. Thus a single DNA insertion event leads to specific tagging of all three Central Dogma molecules - DNA, RNA and Protein.
Molecular tagging methods have been widely used to study protein structure and function and location. Unlike other techniques where the tag sequence is added to cloned cDNA, the CD-tag is added to the complete gene itself thus preserving most of its natural regulation. Our most commonly used CD-tag contains two guest exons, one encoding an epitope tag and the other encoding a GFP (green fluorescent protein) tag. This CD-tag is delivered as a single cassette into our mammalian model cells, the NIH 3T3 mouse fibroblast, by a modified retroviral vector as shown below.
We use a "shotgun" approach to create tagged cell lines that are sorted, purified and characterized. Because the current CD tagging vector uses the GFP as a tag, tagged proteins can be visualized by fluorescence microscopy.
Information about the complete collection of CD-tagged cell lines is available in an online database.
Here are some examples of what mouse NIH 3T3 cells look like in the fluorescence microscope with different genes carrying GFP CD-tags.
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Peter B. Berget
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