Figure 1

Protein tagging system for mammalian cells. Schematic diagram of the tagging strategy. The strategy involves an artificial exon that is delivered into a target cells where it becomes stably associated with the host cell genome. The artificial exon consists of a BSD drug resistance gene sequence flanked by 34-bp loxP sites and lacking an ATG codon. An EGFP coding region lacking translational start and stop codons was placed in-frame with the BSD coding region. The artificial exon contains a branch point sequence, a polypyrimidine-tract sequence, the mandatory AG dinucleotide of the splice acceptor (SA) site and the mandatory GT of the splice donor (SD) site. The presence of a stop codon in the BSD cassette generates a fusion protein containing a truncated version of an endogenous protein fused to the BSD resistance protein and enables selection of BSD-resistant cell clones. Excision of BSD cassette by Cre-mediated recombination leads to the expression of EGFP fused to an endogenous protein. E1, E2 and E3 refer to cellular exons.