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Nucleic Acids Research, 1983, Vol. 11, No. 21 7287-7302
© 1983

MOLECULAR BIOLOGY

Replication and expression in mammalian cells of tramfected DNA; description of an improved erythrocyte ghost fusion technique

F.C. Wiberg1,2, P. Sunnerhagen2, K. Kaltoft1, J. Zeuthen1,3 and G. Bjursell2

1Institute of Human Genetics, Bartholin Building, University of Aarhus DK-8000 Aarhus C, Denmark 2Dept. Medical Biochemistry, University of Goteborg Box 33031, S-400 33 Göteborg, Sweden 3Novo Biomedical, Novo Industri A/S DK-2880 Bagsvaerd, Denmark

Received October 5, 1983. Accepted October 14, 1983.

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 elctron 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 9 form and rolling - circle type were observed.


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