Efficient introduction of plasmid DNA into Trypanosoma brucei and transcription of a transfected chimeric gene.
Electroporation induces efficient transient transfection of Trypanosoma brucei, and the introduced DNA can be transcribed into RNA. When we delivered a high-voltage electric pulse to cells mixed with radiolabeled pBR322, approximately equal to 15% of the plasmid DNA was taken up by the parasites. When transfecting DNA contained a segment of T. brucei ribosomal DNA that included the 5' end of the rRNA gene, the introduced plasmid directed expression of RNA; this RNA expression was shown both by dot blots and by S1 nuclease protection assays carried out under conditions specific for probe hybridization to RNA. In the absence of the ribosomal region, analogous transcription did not occur. We optimized this trypanosomal expression system with regard to electric shock strength, concentration of input DNA, and incubation time after electric shock. This technique enabling specific trypanosome DNA expression in vivo should facilitate the molecular analysis of T. brucei gene expression.
D000818:Animals; D004275:DNA, Ribosomal / Q000235:genetics*; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D010957:Plasmids; D012335:RNA, Ribosomal / Q000235:genetics*; D012337:RNA, Ribosomal, 18S / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection / Q000379:methods; D014346:Trypanosoma brucei brucei / Q000235:genetics*
D004275:DNA, Ribosomal; D012335:RNA, Ribosomal; D012337:RNA, Ribosomal, 18S