GFP as a Genetic Marker Scorable Throughout the Life Cycle of Transgenic Zebra Fish

A fish expression vector, FRM, was constructed by fusing the carp beta-actin promoter and first intron to the ocean pout antifreeze protein terminator and putative boundary element. Mutant forms of the green fluorescent protein (GFP) were engineered into this vector, and the resultant series of vectors, FRMwg, FRM3wg (green GFP), and FRM2bl (blue GFP), were used to make transgenic zebra fish. After microinjection of either supercoiled or linearized DNA into one-celled eggs, GFP-expressing cells could be monitored by fluorescence microscopy commencing with the midblastula transition and continuing through embryogenesis. From adult fish, which retained scorable GFP either as patches or as a uniform fluorescence, 11 green and 1 blue GFP-expressing lines of zebra fish have been established. Expression of GFP was nearly ubiquitous and similar among all of these lines. Embryonic expression could be scored at 15 to 30 hours postfertilization and was seen throughout the embryo with the exceptions of the yolk, red blood cells, and in some lines, portions of the head. Adult expression was in a majority of tissues (e.g., muscle, brain, intestine, and heart, but not red blood cells). The notable difference between lines was that fluorescent eggs were scorable in seven of the lines. Adult homozygotes from a different subset of eight lines could be selected by the relative intensity of the GFP marking when compared with that in sibling heterozygotes. All 12 lines contain apparent single locus, multicopy, tandem integrations (1.5-100 copies per cell) of the transgenic DNA. The fish expression vector FRM could be used to drive nearly ubiquitous and strong expression of gene products other than GFP. The GFP expression vectors, FRMwg, FRM2wg, FRM3wg, and FRM2bl, may be useful for optimization of transgenesis and as a comarker. GFP-expressing zebra fish lines could facilitate experimental analysis of chimerism and in vivo gene targeting.