An in vivo screen for the luciferase transgene in zebrafish.
A simple and economical large-scale in vivo screen for firefly luciferase expression in transgenic zebrafish is described. The screen is a film assay of luminescence during embryogenesis. Either luciferin substrate can be microinjected into the embryo, or the embryo can be raised in a luciferin solution. In a test of transient expression in the G0 (microinjected) generation, a construct with the human cytomegalovirus (CMV) promoter gave higher levels of expression than three other constructs. Using the CMV promoter, injection of supercoiled or linear DNA led to approximately equivalent amounts of expression. Although G0 transient luciferase expression is high enough to be reliably screened, G1 integrated expression is either low or nonexistent, and therefore unscreenable. In the G1 and G2 generations, low-level expression was increased with application of 5-azacytidine. The fact that both transgene methylation and 5-azacytidine activation of expression occurred suggests that methylation is involved in either reducing or eliminating integrated luciferase expression. This in vivo luciferase screen may be useful for insertional mutagenesis, promoter, gene, or enhancer traps, promoter analysis, and optimization of conditions for gene transfer.
Gibbs
P D
PD
Department of Genetics and Cell Biology, Washington State University, Pullman.
Peek
A
A
Thorgaard
G
G
D000818:Animals; D030801:Animals, Genetically Modified; D001374:Azacitidine / Q000494:pharmacology; D015139:Blotting, Southern; D001517:Coleoptera / Q000201:enzymology; D004247:DNA / Q000737:chemistry / Q000235:genetics; D005854:Germ Cells; D008156:Luciferases / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010957:Plasmids; D015027:Zebrafish / Q000235:genetics*
D004247:DNA; D008156:Luciferases; D001374:Azacitidine