The analysis of transient expression in the injected embryos provides a way of checking whether a particular promoter will drive transcription in zebrafish. This allows potential transgenic vectors to be tested before embarking on a major F1 screen. Several viral promoters have been tested in this way. Stuart et al. (9) injected a plasmid containing the SV-40 early region promoter upstream of the Rous sarcoma virus long-terminal repeat (RSV-LTR) fused to the chloram-phenicol acetyltransferase (CAT) gene and showed that CAT activity could be detected as early as 8 h and up to 12 d after injection. This was a complex promoter, but expression appeared to be primarily driven by the RSV-LTR.
Bayer and Campos-Ortega (10) injected two plasmids, one containing the RSV-LTR promoter fused to lacZ, and the second containing a truncated mouse heat-shock promoter linked to lacZ. Injected embryos were fixed and stained for P-glactosidase (P-gal) activity about 24 h later. Embryos injected with the RSV construct exhibited staining in cells of mesodermal origin, whereas the heat-shock construct gave P-gal activity in most tissues.
The most powerful promoter allowing expression in all cell types is probably the immediate early promoter and enhancer of human cytomegalovirus, CMV (11). This promoter fused to lacZ drives expression in most tissues of the zebrafish embryo (12). Gibbs et al. (13) compared the CMV promoter with the SV-40 early promoter and the RSV-LTR promoter, each fused to the luciferase gene. The CMV promoter gave the strongest signal with the lowest concentration of luciferase reaction components, allowing positive fish to be raised to adulthood.
This type of transient assay can be exploited to examine the effect of ectopic expression of one gene on the regulation of a potential downstream gene. The CMV promoter is fused to a full-length cDNA of the first gene, and the plas-mid injected into the embryo. The embryos are subsequently subjected to double in situ hybridization first to locate those cells that are expressing the CMV-driven gene and then to determine whether the presumptive target gene is also active in the same cells. This transient assay is a powerful technique, since it is a quick way of determining potential regulatory cascades and causes far less disruption to the embryo than injecting RNA.
Westerfield et al. (12) used the transient assay to test the ability of mammalian Hox gene promoters to drive the expression of lacZ in specific regions and tissues of the zebrafish. Linear DNA fragments injected at the one cell stage gave expression in one to several hundred cells when examined about a day later. The expression patterns were always mosaic in that expressing cells were mixed with nonexpressing cells in a variegated manner. Mosaicism in these transient assays probably occurs through the uneven distribution and replication of the DNA during subsequent cell divisions and through the failure of some cells to activate the promoter. A similar study has been performed with promoter lacZ fusions for the mammalian GAP-43 gene (14). This approach provides a way of examining the conservation of signaling pathways and cis-acting regulatory elements in evolutionary-related vertebrate genes.
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