Replication and expression in mammalian cells of transfected DNA; description of an improved erythrocyte ghost fusion technique.
After modification of an erythrocyte - ghost fusion technique, DNA can be transferred in large amounts to mammalian cells. The overall recovery permits analysis of transferred DNA immediately after fusion by e.g. Southern - blot hybridization and electron microscopy. In general more than 20% of the cells contain the transfected DNA in their nuclei within 4 hours post fusion. Expression of Polyoma T antigens is delayed, as compared to a normal virus infection, and is detected at about 60 hours after transfer of DNA. During the first days an increase in amount of transferred DNA could be detected which might be due to limited replication. To analyze the replication of hybrid DNA molecules consisting of Polyoma and prokaryotic plasmid DNA sequences, fusion was followed by a normal Polyoma virus infection. The resulting induction of cells to enter the S phase made possible replication analysis of transfected hybrid DNA. Replicating molecules of both the theta form and rolling - circle type were observed.
D000818:Animals; D002459:Cell Fusion; D002460:Cell Line; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009693:Nucleic Acid Hybridization; D011120:Polyomavirus / Q000235:genetics; D051381:Rats; D014158:Transcription, Genetic; D014162:Transfection
D004279:DNA, Viral