Direct gene transfer into Actinidia deliciosa protoplasts: analysis of transient expression of the CAT gene using TLC autoradiography and a GC-MS-based method.
Chloramphenicol acetyl transferase (CAT) gene was used as a reporter gene to assess the conditions for polyethylene glycol (PEG)-mediated transfection of kiwifruit protoplasts. The effect of plasmid concentration and the presence of carrier DNA were each assessed by analysing CAT activity in transfected protoplasts using thin-layer chromatography (TLC) autoradiographic detection of acetylated chloramphenicol. A gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) non-radioactive method was developed for monitoring CAT gene activity. This method provides a high speed of analysis (30 min) and precise means of detecting acetylated products at the nanomolar level, enabling quantification at very low transfection rates. Using this method we optimized plasmid and PEG concentration and also assessed the effect of heat shock on transfection. The best CAT activity was obtained using 30% polyethylene glycol 4000 and by submitting protoplasts to heat shock (45 degrees C, 5 min) prior to transfection.
Oliveira
M M
MM
Departamento de Biologia Vegetal, Faculdade de Ciências de Lisboa, Portugal.
Barroso
J
J
Pais
M S
MS
D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis; D002849:Chromatography, Gas; D002855:Chromatography, Thin Layer; D005638:Fruit / Q000235:genetics*; D008401:Gas Chromatography-Mass Spectrometry; D066298:In Vitro Techniques; D010957:Plasmids; D014162:Transfection
D015500:Chloramphenicol O-Acetyltransferase