Transient expression of pKRT8-LGLR in zebrafish embryos by co-injection of pCMV-CRE or pZP3-CRE. A: Schematic representation of chimeric DNA constructs, pKRT8-LGLR, pCMV-CRE, and pZP3-CRE. B: A 48 hours postfertilization (hpf) embryo injected with pKRT8-LGLR only, showing only GFP expression. C,D: A same 48 hpf embryo co-injection of pKRT8-LGLR and pCMV-CRE, showing both green fluorescent protein (GFP) expression under a blue light (C) and red fluorescent protein (RFP) expression under a yellow light (D). E,F: A same 48 hpf embryo co-injection of pKRT8-LGLR and pZP3-CRE, showing both GFP (E) and RFP (F) expression. Scale bars = 500 μm.

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.

Cre RNA expression in Tg(zp3:cre, krt8:rfp) transgenic zebrafish. A,B: cre RNA expression in 2-72 hours postfertilization (hpf) embryos (A) and in adult transgenic fish (B) as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Embryonic stages in hpf (A) and tissue sources (B) are indicated at the top of each lane. Whole-male transgenic fish (WFB) and selected tissues from female transgenic fish were used for RNA preparation. P, positive control with pZP3-CRE plasmid; N, negative control without template. β-actin probes were used for RT-PCR control. C,D: Ovary expression of cre mRNA in Tg(zp3:cre, krt8:rfp) transgenic female (C) and zp3 mRNA wild-type female (D). Whole-mount in situ hybridization detection was performed with isolated ovary tissues and both cre and zp3 mRNAs were detected in developing oocytes (arrows) but not in the mature oocyte.

Test of Cre-mediated recombination at the chromosome level by breeding of Tg(zp3:cre, krt8:rfp) and Tg(mylz2:loxP-EGFP-loxP)^gz3. A: The 48 hpf embryos with strong, weak, and nil green fluorescent protein (GFP) expression from a cross between female Tg(zp3:cre, krt8:rfp) and male Tg(mylz2:loxP-EGFP-loxP)^gz3. Percentages of each type of embryos based on GFP expression are indicated. B: Polymerase chain reaction (PCR) detection of Cre-mediated recombination in offspring of female double transgenic fish Tg(zp3:cre, krt8:rfp)/Tg(mylz2:loxP-EGFP-loxP)^gz3 and male wild-type fish. In both skin RFP-expressing (cre transgenic) and non-RFP-expressing (cre non-transgenic) embryos (4 days postfertilization), approximately half of them showed Cre-mediated recombination as anticipated from the female germline excision while the other half were loxP non-transgenic individuals. The expected PCR products are 2.1 kb and 1.2 kb, respectively, before and after the Cre-mediated recombination. P, positive PCR control with pMYLZ2-loxP-EGFP-loxP plasmid that is used to generate Tg(mylz2:loxP-EGFP-loxP)^gz3; N, negative PCR control without a template DNA; M, 1-kb DNA ladder. Scale bar = 500 μm.

Activation of loxP-blocked reporter gene expression. A,B: A 48 hours postfertilization (hpf) embryo from a cross between a female Tg(zp3:cre, krt8:rfp) and male Tg(EF:loxP-mCherry-loxP-egfp) to show mCherry (A) and enhanced green fluorescent protein (EGFP; B) expression. C,D: A 48-hpf embryo from a cross between a female wild-type fish and male Tg(EF:loxP-mCherry-loxP-egfp) to show mCherry (C) and GFP (D) expression. E,F: View of mCherry (E) and GFP (F) expression in the ovary from a 1.5-month-old female double transgenic fish Tg(Zp3:Cre, Krt8:RFP)/ Tg(EF:loxp-mCherry-loxp-egfp. G,H: View of mCherry (G) and GFP (H) expression in the ovary from a 1.5-month-old female Tg(EF:loxp-mCherry-loxp-egfp fish. Scale bars = 100 μm.