An artificial promoter construct for heat-inducible misexpression during fish embryogenesis.
Beside spatial distribution, timing of gene expression is a key parameter controlling gene function during embryonic development. Gain-of-function experiments can therefore have quite opposing results, depending on the time of gene activation. Induction techniques are necessary to control timing in these experiments from outside of the organism. Natural heat shock promoters constitute a simple inducible misexpression system, the main disadvantage is a high background level of expression. We present here a new heat stress-inducible bidirectional promoter consisting of multimerized heat shock elements (HSE). The simplified architecture of this promoter largely improves the properties needed for an efficient induction system: dramatically reduced background activity, improved inducibility, and loss of all tissue specific components. Based on this new artificial promoter, we present a transient induction system for fish embryos. Application of this new induction system for Fgf8 misexpression during embryonic development reveals different windows of competence during eye development. A dramatic early phenotype resulting in loss of the eyes is observed for conventional mRNA injection. Later activation, by using our inducible promoter, uncovers different eye phenotypes like cyclopic eyes. Even after 14 days, an efficient heat stress response could be evoked in the injected embryos. The HSE promoter therefore represents a new artificial heat shock promoter with superior properties, making possible transient experiments with inducible misexpression at various stages of development.
Bajoghli
Baubak
B
Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria.
Aghaallaei
Narges
N
Heimbucher
Thomas
T
Czerny
Thomas
T
D000818:Animals; D030801:Animals, Genetically Modified; D003001:Cloning, Molecular / Q000379:methods*; D051524:Fibroblast Growth Factor 8; D005346:Fibroblast Growth Factors / Q000235:genetics* / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins; D006360:Heat-Shock Proteins / Q000235:genetics*; D006358:Hot Temperature; D006801:Humans; D008156:Luciferases; D008164:Luminescent Proteins; D051379:Mice; D008845:Microinjections; D009990:Oryzias / Q000196:embryology* / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013997:Time Factors; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
C496456:FGF8 protein, human; D006360:Heat-Shock Proteins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D051524:Fibroblast Growth Factor 8; D005346:Fibroblast Growth Factors; D008156:Luciferases