Detection of transcription and translation in situ with biotinylated molecular probes in cells transfected with recombinant DNA plasmids.
The efficiency of transfection and subsequent expression of recombinant DNA plasmids in monolayers of CV-1 monkey kidney cells was analyzed by immunoperoxidase and in situ hybridization with biotin-nucleotide-labeled DNA molecular probes. Two recombinant plasmids were used for transfection. Both contained the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV) as the transcriptional promoter, but two different coding sequences were employed [bacterial chloramphenicol acetyltransferase (pRSVcat) and mouse casein alpha (pRSVcsn alpha)]. In our experiments up to 25% of the transfected cells were positive for pRSVcat expression by indirect immunoperoxidase assay with affinity-purified, biotinylated anti-goat gamma-globulin after exposure to goat anti-chloramphenicol acetyltransferase antibody. In duplicate cultures, where pRSVcat expression was monitored by in situ hybridization signal that was restricted to the cytoplasm in positive cells was identified as pRSVcat RNA by its sensitivity to alkali. Although transfection of CV-1 cells with pRSVcsn-alpha did not result in immunologically detectable alpha casein, greater than 14% of the cells possessed cytoplasmic RNA concentrations detectable by in situ hybridization. These observations provide comparative information on in situ hybridization and immunoperoxidase techniques. They further indicate that in situ hybridization can be used to evaluate the effectiveness of transfection with recombinant expression vectors.
D000123:Acetyltransferases / Q000235:genetics; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D001710:Biotin; D002364:Caseins / Q000235:genetics; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase; D002522:Chlorocebus aethiops; D004274:DNA, Recombinant; D007124:Immunoenzyme Techniques; D007668:Kidney; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D014176:Protein Biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection
D002364:Caseins; D004274:DNA, Recombinant; D001710:Biotin; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase