(A) Map of EF-lacZ. The EF1α enhancer-promoter is shown as a large open box, the lacZ gene is cross-hatched, and the plasmid vector pSP72 is shown as a line. The relative locations of the polymerase chain reaction (PCR) primers used to identify transgenic fish are indicated by arrows. (B) Identification of germline transgenic fish by PCR. Genomic DNA was extracted from a pool of embryos and was used in a PCR reaction with two pairs of primers. The first pair of primers is specific to the injected plasmid as shown in (A). The second pair of primers is specific to zebrafish homeobox gene, ZF-2.1 (Njolstad et al., 1988), generating a 475-bp product which is present in all the lines. Lane 1 is a 100-bp ladder. Lanes 2-9 are from pools of embryos from founder fish injected with EF-lacZ BglII fragment. Lane 10 is a positive control containing DNA from uninjected fish mixed with 20pg EF-lacZ plasmid. Lane 11 is a negative control containing DNA from uninjected fish. The founder fish in lane 5 (EF-50F) was detected as positive for the transgene.

Expression of lacZ in F1 transgenic embryos. F1 embryos from line EF-head were fixed at the early gastrula and stained with X-Gal. Three embryos which express lacZ show readily discernible blue staining (A). Expression patterns of lines EF-blue (B), EF-head (C), EF-patchy (D), and EF-neuron (E) are shown at different developmental stages. Expression of lacZ in embryos from line EF-neuron is restricted to the CaP motoneurons in the spinal cord (F, G, and H). sc, spinal cord. n, notochord. The distribution of lacZ-expressing CaP cells is variable between embryos as shown from the dorsal view (I).

lacZ expression in live embryos detected by FDG staining. F1 embryos of line EF-blue were stained with FDG as described under Materials and Methods. Two FDG-positive embryos are shown with two FDG-negative embryos (A). These embryos were then fixed and stained individually with X-Gal. (B) The FDG-positive embryos are also X-Gal-positive and FDG-negative embryos are X-Gal-negative.