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-acti n promoter and first intron to the ocean pout antifreeze protein terminator and putative boundary element. Mutant forms of the green fluorescent prote in (GFP) were engineered into this vector, and the resultant series of vect ors, FRMwg, FRM3wg (green GFP), and FRM2bl (blue GFP), were used to make tr ansgenic zebra fish. After microinjection of either supercoiled or lineariz ed DNA into one-celled eggs, GFP-expressing cells could be monitored by flu orescence microscopy commencing with the midblastula transition and continu ing through embryogenesis. From adult fish, which retained scorable GFP eit her as patches or as a uniform fluorescence, 11 green and 1 blue GFP-expres sing lines of zebra fish have been established. Expression of GFP was nearl y ubiquitous and similar among all of these lines. Embryonic expression cou ld be scored at 15 to 30 hours postfertilization and was seen throughout th e embryo with the exceptions of the yolk, red blood cells, and in some line s, 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 notab le difference between lines was that fluorescent eggs were scorable in seve n of the lines. Adult homozygotes from a different subset of eight lines co uld be selected by the relative intensity of the GFP marking when compared with that in sibling heterozygotes. All 12 lines contain apparent single lo cus, multicopy, tandem integrations (1.5-100 copies per cell) of the transg enic DNA. The fish expression vector FRM could be used to drive nearly ubiq uitous and strong expression of gene products other than GFP. The GFP expre ssion vectors, FRMwg, FRM2wg, FRM3wg, and FRM2bl, may be useful for optimiz ation of transgenesis and as a comarker. GFP-expressing zebra fish lines co uld facilitate experimental analysis of chimerism and in vivo gene targetin g.