Fig. 2

PCR and Southern hybridization analyses of F₁ fish. (A) PCR analyses of genomic DNA extracted from caudal fin clippings of F₁ fish from fkk-1 and fkk-7. Primer pairs used for PCR and the sizes of the PCR products are shown on the right. Two (fkk-1, lanes 1 and 2) and 25 F₁ fish were found positive for PCR using Tol2f5/Tol2r7 out of 68 and 50 F₁ fish from fkk-1 and fkk-7, respectively. PCR analyses of the 25 F₁ fish from fkk-7 yielded the same results, and four representatives are shown (fkk-7, lanes 1-4). The same four samples were used also for Southern hybridization (described in C). In the control (P), 100 ng of zebrafish genomic DNA plus 1 pg of the (Tol2-tyr)ΔRV plasmid DNA were used as a PCR template. (B) Southern hybridization analysis of F₁ fish from fkk-1. DNA samples of two fish (A) were digested with either EcoRV or EcoRI and hybridized with the probe shown in Fig. 1. (C) Southern hybridization analysis of F₁ fish from fkk-7. DNA samples of four fish (A) were digested with either EcoRV or EcoRI and hybridized with the probe (Fig. 1). EcoRI bands of four different sizes, shown as a-d on the right, correspond to the fkk-7a, fkk-7b, fkk-7c, and fkk-7d insertions described in the text. The lower bands in EcoRV lane 1 are probably a triplet. (D) Southern hybridization analysis of F₂ fish from the lane 1 F₁ fish (fkk-7). Ten fish out of 14 F₂ fish, which were positive for PCR using Tol2f5 and Tol2r7, were analyzed. DNA samples were digested with EcoRI and hybridized with the probe (Fig. 1). Fish with fkk-7b alone (lanes 1, 2, 10), fkk-7b alone (lanes 4, 6, 8), and both fkk-7b and fkk-7c (lanes 3, 5, 7, 9) were identified.