Fig. 2

Co-existence of red fluorescent protein (RFP) expression and cre DNA in Tg(zp3:cre, krt8:rfp) F1 embryos. A: Skin-specific RFP expression in Tg(zp3:cre, krt8:rfp) embryos: 24 hours postfertilization (hpf; upper) and 72 hpf (lower). B: Presence of cre DNA in RFP-positive embryos. Genomic DNAs were prepared from 5 days postfertilization embryos individually and cre DNA was detected by polymerase chain reaction (PCR). The picture shows nine RFP-positive embryos and one RNA-negative embryo. P, positive PCR control with pZP3-CRE template; N, negative PCR control without template; M, 100-bp DNA marker (Promega). C: Demonstration of the Cre-mediated excision of floxed egfp in Tg(zp3:cre, krt8:rfp) transgenic fish. PCR was performed for both RFP-positive and -negative embryos using primers P1 and R1 (upper) as indicated in the diagram with expected sizes of PCR products before and after Cre-mediated excision. Presence of cre DNA was also amplified by PCR using a pair of cre-specific primers (lower). The 850-bp recombined fragment and cre DNA are co-existed in skin RFP expressing embryos (RFP+) but not in the non-RNA expressing embryos (RFP-). Primers P1+G1 amplified 750-bp unexcised gfp fragment. P, positive PCR control with templates pKRT8-LGLR (upper) or pZP3-CRE (lower); N, negative PCR control without template; M, 1-kb DNA marker. In the diagram, LoxP, EGFP, and RFP DNAs are indicated by blue triangles, green and red boxes, respectively. Scale bars = 500 μm.