PCR amplification of the mariner element in F₁ progeny. (a) Genomic DNA was isolated from pools of 20-30 F₁ generation larvae from six pairwise matings of founder fish (numbered 1-6) and used as template for amplification of mariner sequences. Nine of the pools of larvae from four different crosses tested positive for the transgene as indicated by a product at 450 bp (arrow). (b) Analysis of DNA samples isolated from fin clippings of individual F₁ offspring from mating of founder pair number 5. Genomic DNA from 2 of the 15 individuals demonstrated the presence of mariner sequences (arrow) (lane M, 100-bp marker; Promega; lane B, no DNA control; lane C, positive control containing 1 ng pWE1 plasmid).

PCR demonstration of inheritance of the mariner element without plasmid vector sequences. (a) Genomic DNA isolated from individual F₂ larvae was tested for the presence of the mariner element and flanking plasmid sequences. Amplification of mariner sequences identified 11 of 16 (68%) larvae as positive for the transgene (arrow) (lane M, 100-bp marker, Promega; lane B, no DNA control). (b) Amplification with one primer specific for mariner and one for the M13 reverse sequence flanking the insert in the pWE1 plasmid revealed none possessed this plasmid DNA. The control lane (C), containing 1 ng pWE1 plasmid, demonstrated the expected amplification product of 680 bp (arrowhead).

Evidence for transposition of the peach element by Southern blot analysis. Genomic DNA from lines 1085 (a), 1060 (b) and 1061 (c) were digested with BamHI (B), EcoRI (E), and SacI (S), and filters were probed with the 1.3-kb peach element from pWE1. If the peach element had integrated with flanking plasmid sequences, then digestion with EcoRI should generate a single 1.3-kb band, and digestion with SacI, which recognizes one site in the peach element and one in the multicloning site of pWE1, should produce a fragment of 800 bp and a second fragment with a size dependent upon the site of integration. However, the hybridization patterns for the three samples differ, and the fragments are much larger than expected. These findings indicate that integration of the peach element occurred without flanking plasmid sequences and that the genomic sites of integration are different. (Molecular weight standards are shown to the left. The arrow indicates the location of the intact 1.3-kb peach element from pWE1 following digestion with EcoRI.)