Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters.
Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole-mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker-mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2-kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5-kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8-kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters.
Ju
B
B
Department of Biological Sciences, National University of Singapore, Singapore.
Xu
Y
Y
He
J
J
Liao
J
J
Yan
T
T
Hew
C L
CL
Lam
T J
TJ
Gong
Z
Z
D000818:Animals; D030801:Animals, Genetically Modified; D003402:Creatine Kinase / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D017403:In Situ Hybridization; D007527:Isoenzymes; D007633:Keratins / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics*; D008957:Models, Genetic; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D012269:Ribosomal Proteins / Q000235:genetics; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics*
D018076:DNA, Complementary; D007527:Isoenzymes; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; D049452:Green Fluorescent Proteins; D007633:Keratins; D003402:Creatine Kinase