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Nucleic Acids Research, 1985, Vol. 13, No. 15 5545-5561
© 1985

Articles

Fate of exogenous recombinant plasmids introduced into mouse and human cells

Giuseppe Biamonti, Giuliano Della Valle1, Daniela Talarico1, Fabio Cobianchi, Silvano Riva and Arturo Falaschi*

Istituto di Genetica Biochimica ed Evoluzionistica del C.N.R. Via Abbiategrasso, 207-27100 Pavia 1Dipartmentimento di Genetica e Microbiologia, Universitá di Pavia Via S.Epifanio, 14-27100 Pavia, Italy

*To Whom correspondence should be addressed

Received April 22, 1985. Revised June 28, 1985. Accepted July 10, 1985.

We have constructed a number of plasmids selectable in both E. coli and mouse or human cells. Human DNA sequences were inserted and the recombinant plasmids were used to transfect either mouse or human cells by the Ca-phosphate precipitation technique. We have observed that: (i) competent cells uptake large amounts of plasmid DNA; (ii) input plasmids persist in trasformed mammalian cells as free unreplicating circular molecules for up to 20 generations; such persistence does not depend on the presence of selective markers; (iii) plasmids incorporated into mouse L-cells undergo widespread rearrangements (in the absence of replication) entailing mostly deletions of both human and bacterial sequences which yield smaller products; the latter appear to be more stable in a subsequent transformation cycle. Surprisingly such rearrangements are almost totally absent in transformed human KB-cells. This property of human KB-cells mayprove useful for the development of a vector apt at cloning and expressing human DNA sequences. Unlike what has been observed in yeast, no "autonomously replicating sequence" can be detected in mammalian cells by randomly cloning human DNA sequences into a selectable plasmid and screening for an increased transformation efficiency.


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