An assay for transient gene expression in transfected Drosophila cells, using [3H]guanine incorporation.
We have developed an assay for transient gene expression using a dominant-selectable marker previously employed to transform Drosophila cultured cells. Drosophila hydei cells transfected with a functional Escherichia coli xanthine guanine phosphoribosyl transferase gene (gpt), under the control of the long terminal repeats (LTRs) of the copia transposable element, rapidly incorporate guanine into acid-precipitable counts. Autoradiographic analysis in situ shows that approximately 20% of cells take up, and express, the gpt gene. This transient gpt expression depends on the Drosophila promoter sequences since vectors with the gpt gene in reverse orientation to the copia LTRs fail to incorporate guanine. Deletion analysis confirms that the LTRs are essential for gpt gene expression. Similarly, cells transfected with gpt controlled by the Drosophila 70 000 mol. wt. heat-shock (hsp 70) promoter show regulated guanine incorporation when heat shocked. The efficiency of the copia LTRs varies considerably between the cell lines we tested, whereas that of the hsp 70 promoter does not. The heterologous promoters of the Rous sarcoma virus (RSV) and simian virus 40 (SV40) function poorly in these cells.
D000818:Animals; D002460:Cell Line; D004262:DNA Restriction Enzymes; D004330:Drosophila / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D005799:Genes, Dominant; D005822:Genetic Vectors; D006147:Guanine / Q000378:metabolism; D010430:Pentosyltransferases / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D014162:Transfection; D014316:Tritium
D014316:Tritium; D006147:Guanine; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase; D004262:DNA Restriction Enzymes