Use of an IRES bicistronic construct to trace expression of exogenously introduced mRNA in zebrafish embryos

To understand gene function in developing vertebrate embryos, co-injection of an mRNA for a reporter protein and an mRNA for a testing factor is widel y used. However; because of the mosaic segregation of injected nucleic acid s during early embryogenesis, whether both mRNAs are translated in the same cell remains uncertain. In the present study, we tested a new system of tr acing the expression of a testing gene in zebrafish using an internal ribos omal entry site (IRES) to express two proteins from the same mRNA template, thus eliminating the problem of independent translation observed in co-inj ection essays. A DNA construct was made for synthesizing bicistronic mRNA f or NeuroD, a neurogenic transcription factor; and the enhanced green fluore scent protein (EGFP) reporter When the bicistronic mRNA for NeuroD and EGFP was injected into zebrafish embryos at one cell stage, all EGFP-expressing embryos showed ectopic expression of neuroD mRNA and the mRNA of its poten tial downstream gene, islet-l. Thus, the IRES bicistronic mRNA construct mi ght be a more convincing means of analyzing gene function in developing zeb rafish embryos.