Characterizations of Hirudo medicinalis DNA promoters for targeted gene expression.
The expression of exogenous genes in neurons and other cells has become a powerful means for studying the function of encoded proteins. We report here on the isolation and functional analysis of three Hirudo medicinalis actin gene promoters and the 5' UTR of a leech elongation factor-1alpha (HmEF-1alpha) gene. In situ hybridization labeling revealed that the EF-1alpha gene and one of the actins had pan-neuronal expression, whereas, the other two actin genes were expressed by the embryo's body wall musculature. Comparative analysis shows that they all display many features typical of actin and EF-1alpha promoters from other species, including canonical TATA box sequences and predicted general transcription factor binding sites (such as CCATT, CarB boxes and CG-rich regions). The ability of these 5' UTR sequences to drive expression of the enhanced green fluorescent protein (EGFP), leech cytoplasmic actin and leech synaptobrevin was examined. Direct intracellular nuclear, but not cytoplasmic, microinjection of each of the promoter sequences was found to produce reliably cellular expression of the reporter construct in both neuronal and muscle cells. These results introduce reliable and effective methods to selectively express genes in individual cells of the leech in vivo during embryonic development.
Baker
Michael W
MW
Section of Cell and Developmental Biology, Division of Biology, University of California, San Diego, La Jolla, CA 92093, USA. mwbaker@ucsd.edu
Macagno
Eduardo R
ER
D020121:5' Untranslated Regions / Q000235:genetics; D000199:Actins / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002467:Cell Nucleus / Q000235:genetics; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics*; D004625:Embryo, Nonmammalian / Q000502:physiology; D015870:Gene Expression / Q000502:physiology*; D018390:Gene Targeting / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D047709:Hirudo medicinalis / Q000235:genetics*; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014018:Tissue Distribution; D014162:Transfection
D020121:5' Untranslated Regions; D000199:Actins; D020648:Peptide Elongation Factor 1; D049452:Green Fluorescent Proteins; D004247:DNA