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. 1981 Nov 25;9(22):6199–6217. doi: 10.1093/nar/9.22.6199

Recovery of recombinant bacterial plasmids from E. coli transformed with DNA from microinjected mouse cells.

P J Kretschmer, A H Bowman, M H Huberman, L Sanders-Haigh, L Killos, W F Anderson
PMCID: PMC327594  PMID: 6273826

Abstract

We have previously described the isolation of thymidine kinase positive (TK+), human beta-globin gene-containing colonies following co-microinjection of mouse TK- L cells with two recombinant pBR322 plasmids, one containing the TK gene of herpes simplex virus type I (plasmid pXl), and the second containing a human genomic DNA fragment within which is the human beta-globin gene (plasmid pRKl). DNA isolated from one such clone was used in bacterial transformation experiments with a selection for tetracycline-resistant colonies (that is, for cells containing pRKl). A total of forty-two tetracycline-resistant colonies were isolated, thirty of which contained circular pRK1 molecules identical to those originally injected. The remaining twelve colonies contained unique plasmids that were grouped into five different classes of recombinant molecules. All five of these unique recombinant classes appear to contain a common deletion endpoint occurring at a specific region of the pBR322 segment of pRKl. Four of the unique recombinant classes appear to have arisen from the deletion of a segment of a pRKl trimer or dimer molecule, while the fifth class appears to have resulted from recombination between pRKl and pXl followed by a deletion event within this recombinant. It is uncertain whether these deletions are occurring within the eukaryotic cell or upon subsequent transformation of the bacterial cell. If the latter, then the passage of the plasmid DNA through the eukaryotic cell alters a specific site of the pBR322 DNA in such a way that deletions can occur at a high frequency in this region when the plasmid DNA is introduced back into a bacterial cell. Thus, we have established a prokaryote-eukaryote-prokaryote DNA transfer and recovery system which should be useful in studies on DNA replication and the regulation of gene expression in higher eukaryotes.

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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. Bacchetti S., Graham F. L. Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1590–1594. doi: 10.1073/pnas.74.4.1590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beggs J. D. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978 Sep 14;275(5676):104–109. doi: 10.1038/275104a0. [DOI] [PubMed] [Google Scholar]
  4. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  5. Capecchi M. R. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell. 1980 Nov;22(2 Pt 2):479–488. doi: 10.1016/0092-8674(80)90358-x. [DOI] [PubMed] [Google Scholar]
  6. Chang A. C., Lansman R. A., Clayton D. A., Cohen S. N. Studies of mouse mitochondrial DNA in Escherichia coli: structure and function of the eucaryotic-procaryotic chimeric plasmids. Cell. 1975 Oct;6(2):231–244. doi: 10.1016/0092-8674(75)90014-8. [DOI] [PubMed] [Google Scholar]
  7. Cline M. J., Stang H., Mercola K., Morse L., Ruprecht R., Brown J., Salser W. Gene transfer in intact animals. Nature. 1980 Apr 3;284(5755):422–425. doi: 10.1038/284422a0. [DOI] [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. Enquist L. W., Vande Woude G. F., Wagner M., Smiley J. R., Summers W. C. Construction and characterization of a recombinant plasmid encoding the gene for the thymidine kinase of Herpes simplex type 1 virus. Gene. 1979 Nov;7(3-4):335–342. doi: 10.1016/0378-1119(79)90052-0. [DOI] [PubMed] [Google Scholar]
  10. Hamer D. H., Smith K. D., Boyer S. H., Leder P. SV40 recombinants carrying rabbit beta-globin gene coding sequences. Cell. 1979 Jul;17(3):725–735. doi: 10.1016/0092-8674(79)90279-4. [DOI] [PubMed] [Google Scholar]
  11. Hanahan D., Lane D., Lipsich L., Wigler M., Botchan M. Characteristics of an SV40-plasmid recombinant and its movement into and out of the genome of a murine cell. Cell. 1980 Aug;21(1):127–139. doi: 10.1016/0092-8674(80)90120-8. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Hsiao C. L., Carbon J. High-frequency transformation of yeast by plasmids containing the cloned yeast ARG4 gene. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3829–3833. doi: 10.1073/pnas.76.8.3829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hsiung N., Warrick H., deRiel J. K., Tuan D., Forget B. G., Skoultchi A., Kucherlapati R. Cotransfer of circular and linear prokaryotic and eukaryotic DNA sequences into mouse cells. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4852–4856. doi: 10.1073/pnas.77.8.4852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Huttner K. M., Scangos G. A., Ruddle F. H. DNA-mediated gene transfer of a circular plasmid into murine cells. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5820–5824. doi: 10.1073/pnas.76.11.5820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kaufman R. E., Kretschmer P. J., Adams J. W., Coon H. C., Anderson W. F., Nienhuis A. W. Cloning and characterization of DNA sequences surrounding the human gamma-, delta-, and beta-globin genes. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4229–4233. doi: 10.1073/pnas.77.7.4229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kretschmer P. J., Kaufman R. E., Coon H. C., Chen M. J., Geist C. E., Nienhuis A. W. Hemoglobin switching in sheep. Cloning and characterization of the beta A and beta-like embryonic globin genes from genomic DNA. J Biol Chem. 1980 Apr 10;255(7):3204–3211. [PubMed] [Google Scholar]
  18. Lawn R. M., Fritsch E. F., Parker R. C., Blake G., Maniatis T. The isolation and characterization of linked delta- and beta-globin genes from a cloned library of human DNA. Cell. 1978 Dec;15(4):1157–1174. doi: 10.1016/0092-8674(78)90043-0. [DOI] [PubMed] [Google Scholar]
  19. Lusky M., Botchan M. Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences. Nature. 1981 Sep 3;293(5827):79–81. doi: 10.1038/293079a0. [DOI] [PubMed] [Google Scholar]
  20. Maitland N. J., McDougall J. K. Biochemical transformation of mouse cells by fragments of herpes simplex virus DNA. Cell. 1977 May;11(1):233–241. doi: 10.1016/0092-8674(77)90334-8. [DOI] [PubMed] [Google Scholar]
  21. Meselson M., Yuan R. DNA restriction enzyme from E. coli. Nature. 1968 Mar 23;217(5134):1110–1114. doi: 10.1038/2171110a0. [DOI] [PubMed] [Google Scholar]
  22. Mulligan R. C., Howard B. H., Berg P. Synthesis of rabbit beta-globin in cultured monkey kidney cells following infection with a SV40 beta-globin recombinant genome. Nature. 1979 Jan 11;277(5692):108–114. doi: 10.1038/277108a0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wagner M. J., Sharp J. A., Summers W. C. Nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1441–1445. doi: 10.1073/pnas.78.3.1441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wigler M., Silverstein S., Lee L. S., Pellicer A., Cheng Y. c., Axel R. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell. 1977 May;11(1):223–232. doi: 10.1016/0092-8674(77)90333-6. [DOI] [PubMed] [Google Scholar]

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