Fig. 2

Identification of germ-line transgenic fish by PCR. Genomic DNA was extracted from a pool of embryos from a single mating and was used in two PCR reactions that were run in adjacent lanes on a 2% agarose gel: samples 1-4 test genomic DNAs from embryo pools of four fish injected with pRSV-βGal, and samples 5-8 test pools from four pCH110-injected animals. The first lane in each pair is a positive control reaction containing primers from the zebrafish homeobox gene, ZF-21 (19), generating a 500-bp product; the second contains primers specific to the injected plasmids. The control lanes contain DNA from uninjected fish. The first control lane contains ZF21 primers, and the second, third, and fourth lanes contain both sets of primers directed to the injected plasmids. The third and fourth control lanes also contain 100 fg of the appropriate plasmid. Primers for fish injected with pRSV-/βGal generate a 1.2-kb product (third control lane), and the primers directed to pCH110 generate a 0.6-kb product (fourth control lane). Samples 1 and 4 demonstrate PCR amplification of the RSV-lacZ sequences and consequent germ-line transmission in these fish. Lane M: Hae III-digested OX174 size markers.