Epitope Tagging Genomic DNA Using a CD-Tagging Tn10 Minitransposon

Here, we describe an efficient system for epitope tagging cloned genes by CD tagging using a mini-Tn10 transposon delivery vector. The system was tested against a λFIX genomic clone of the human nucleolin gene. Transfection of HeLa cells with the tagged gene led to the expression of both the appropriately spliced tagged transcript and the appropriately localized tagged protein

CD-Tagging: A New Approach to Gene and Protein Discovery and Analysis

We describe a new method for gene discovery and analysis, CD-tagging, that puts specific molecular tags on a gene, its transcript and its protein product. The method has been successfully tested in two organisms, the haploid unicellular alga Chlamydomonas reinhardtiiand the metazoan Drosophila melanogaster. The method utilizes a specially designed DNA molecule, the CDcassette, that contains splice acceptor and donor sites surrounding a short open reading frame. Insertion of the CD-cassette into an intron in a target gene introduces a new exon, represented by the open reading frame of the CD-cassette, surrounded by two functional hybrid introns. As a result (i) the gene is tagged by a specific nucleotide sequence, (ii) the mRNA is tagged by a specific nucleotide sequence and (iii) the protein is tagged by a specific peptide sequence. Because these tags are unique, specific nucleotide or antibody probes can be used to obtain and/or analyze the gene, transcript or protein. As a gene discovery technology, CD-tagging has two unique advantages: 1) Genes can be identified through a primary screen at the protein level, and so the very process by which a gene is identified provides specific empirical information about its biological function. 2) The cassette arms, which are spliced out of the transcript of the target gene, are available to carry a wide variety of DNA sequences, such as genes encoding drug resistance that can be used to select for the presence of the CD-cassette in the genome