Principle of in vivo epitope-tagging method. (A) The concept of the method is schematically shown. The mini-exon consisting of a synthetic myc-epitope sequence flanked by splice-acceptor and -donor sequences is integrated into an intron of a gene as a single copy. This permits the incorporation of an epitope-tag sequence within a protein coding sequences. Secreted proteins can be detected by ELISA using 9E10 antibody, a commonly used antibody against the myc-epitope sequence . Intracellular protein localization can be detected by immunostaining method using 9E10 antibody. (B) "EpiTag" vector and its mode of operation. EpiTag vector consists of a puromycin-resistant gene cassette flanked by a pair of loxP sequences that preceeds the mini-exon cassette. The puromycin-resistant cassette consists of a synthetic splice acceptor sequence followed by internal ribosomal entry sequence (IRES) fused to the puromycin-resistant sequence that is followed by poly-adenylation sequences. When this vector integrates into an intron, the endogenous protein is expressed as a truncated form but the puromycin-resistant marker is also expressed using the second translation start site in IRES. As a consequence, the cells survive in puromycin containing media. When the puromycin-resistant cassette is excised by Cre-recombinase, the protein is expressed as a full length and as a myc-epitope tagged form. Unique restriction enzyme site, XbaI, in the construct is indicated. The probe used for Southern blot analysis is also indicated. (C) Southern blot analysis to determine the number of integration events of EpiTag transgene per single cell (clone). Results from ten independent clones are shown. The XbaI-digested DNA was analyzed. Single-band indicates a single-site integration event. Multiple-bands indicate a single-copy but a multiple-site integration event. The clone numbers are indicated at the top and the ones that show a multiple-site integration event are indicated with (*). This result indicates approximately 80% of the clones represent single-site integration events of the transgene. S7B2: beta-actin; L1E4:beta ATP synthase; S6E11:CD44 antigen; S5F3:dual specificity protein phosphatase 5; L1A9:enolase alpha; S5A9:integrin alpha6; L2C11:phosphate carrier precursor isoform 1b; F1A5:similar to prokaryotic-type class I peptide chain release factors; S6G11:ribosomal protein L3; F1A3:40S ribosomal protein S12; S5E5:40S ribosomal protein S19.