Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1985 Aug 12;13(15):5545–5561. doi: 10.1093/nar/13.15.5545

Fate of exogenous recombinant plasmids introduced into mouse and human cells.

G Biamonti, G Della Valle, D Talarico, F Cobianchi, S Riva, A Falaschi
PMCID: PMC321889  PMID: 4034393

Abstract

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 transformed 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 may prove 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.

Full text

PDF
5545

Images in this article

5552Image
on p.5552
5554Image
on p.5554
5555Image
on p.5555

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson W. F., Killos L., Sanders-Haigh L., Kretschmer P. J., Diacumakos E. G. Replication and expression of thymidine kinase and human globin genes microinjected into mouse fibroblasts. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5399–5403. doi: 10.1073/pnas.77.9.5399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Binétruy B., Meneguzzi G., Breathnach R., Cuzin F. Recombinant DNA molecules comprising bovine papilloma virus type 1 DNA linked to plasmid DNA are maintained in a plasmidial state both in rodent fibroblasts and in bacterial cells. EMBO J. 1982;1(5):621–628. doi: 10.1002/j.1460-2075.1982.tb01218.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Breitman M. L., Tsui L. C., Buchwald M., Siminovitch L. Introduction and recovery of a selectable bacterial gene from the genome of mammalian cells. Mol Cell Biol. 1982 Aug;2(8):966–976. doi: 10.1128/mcb.2.8.966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Calos M. P., Lebkowski J. S., Botchan M. R. High mutation frequency in DNA transfected into mammalian cells. Proc Natl Acad Sci U S A. 1983 May;80(10):3015–3019. doi: 10.1073/pnas.80.10.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  7. Colbere-Garapin F., Chousterman S., Horodniceanu F., Kourilsky P., Garapin A. C. Cloning of the active thymidine kinase gene of herpes simplex virus type 1 in Escherichia coli K-12. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3755–3759. doi: 10.1073/pnas.76.8.3755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dagert M., Ehrlich S. D. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 1979 May;6(1):23–28. doi: 10.1016/0378-1119(79)90082-9. [DOI] [PubMed] [Google Scholar]
  9. DiMaio D., Treisman R., Maniatis T. Bovine papillomavirus vector that propagates as a plasmid in both mouse and bacterial cells. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4030–4034. doi: 10.1073/pnas.79.13.4030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  11. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  12. Huberman J. A., Riggs A. D. On the mechanism of DNA replication in mammalian chromosomes. J Mol Biol. 1968 Mar 14;32(2):327–341. doi: 10.1016/0022-2836(68)90013-2. [DOI] [PubMed] [Google Scholar]
  13. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. doi: 10.1007/BF00267254. [DOI] [PubMed] [Google Scholar]
  15. Loyter A., Scangos G. A., Ruddle F. H. Mechanisms of DNA uptake by mammalian cells: fate of exogenously added DNA monitored by the use of fluorescent dyes. Proc Natl Acad Sci U S A. 1982 Jan;79(2):422–426. doi: 10.1073/pnas.79.2.422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pellicer A., Robins D., Wold B., Sweet R., Jackson J., Lowy I., Roberts J. M., Sim G. K., Silverstein S., Axel R. Altering genotype and phenotype by DNA-mediated gene transfer. Science. 1980 Sep 19;209(4463):1414–1422. doi: 10.1126/science.7414320. [DOI] [PubMed] [Google Scholar]
  17. Perucho M., Hanahan D., Wigler M. Genetic and physical linkage of exogenous sequences in transformed cells. Cell. 1980 Nov;22(1 Pt 1):309–317. doi: 10.1016/0092-8674(80)90178-6. [DOI] [PubMed] [Google Scholar]
  18. Razzaque A., Chakrabarti S., Joffee S., Seidman M. Mutagenesis of a shuttle vector plasmid in mammalian cells. Mol Cell Biol. 1984 Mar;4(3):435–441. doi: 10.1128/mcb.4.3.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Razzaque A., Mizusawa H., Seidman M. M. Rearrangement and mutagenesis of a shuttle vector plasmid after passage in mammalian cells. Proc Natl Acad Sci U S A. 1983 May;80(10):3010–3014. doi: 10.1073/pnas.80.10.3010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. SZYBALSKA E. H., SZYBALSKI W. Genetics of human cess line. IV. DNA-mediated heritable transformation of a biochemical trait. Proc Natl Acad Sci U S A. 1962 Dec 15;48:2026–2034. doi: 10.1073/pnas.48.12.2026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Scangos G. A., Huttner K. M., Juricek D. K., Ruddle F. H. Deoxyribonucleic acid-mediated gene transfer in mammalian cells: molecular analysis of unstable transformants and their progression to stability. Mol Cell Biol. 1981 Feb;1(2):111–120. doi: 10.1128/mcb.1.2.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Scangos G., Ruddle F. H. Mechanisms and applications of DNA-mediated gene transfer in mammalian cells - a review. Gene. 1981 Jun-Jul;14(1-2):1–10. doi: 10.1016/0378-1119(81)90143-8. [DOI] [PubMed] [Google Scholar]
  23. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  24. Spandidos D. A., Harrison P. R., Paul J. Replication and amplification of recombinant plasmid molecules as extra chromosomal elements in transformed mammalian cells. Exp Cell Res. 1982 Sep;141(1):149–158. doi: 10.1016/0014-4827(82)90077-5. [DOI] [PubMed] [Google Scholar]
  25. Tsui L. C., Breitman M. L., Siminovitch L., Buchwald M. Persistence of freely replicating SV40 recombinant molecules carrying a selectable marker in permissive simian cells. Cell. 1982 Sep;30(2):499–508. doi: 10.1016/0092-8674(82)90247-1. [DOI] [PubMed] [Google Scholar]
  26. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wieslander L. A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels. Anal Biochem. 1979 Oct 1;98(2):305–309. doi: 10.1016/0003-2697(79)90145-3. [DOI] [PubMed] [Google Scholar]
  28. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES