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. 1997 Oct;179(19):6076–6083. doi: 10.1128/jb.179.19.6076-6083.1997

Convenient and reversible site-specific targeting of exogenous DNA into a bacterial chromosome by use of the FLP recombinase: the FLIRT system.

L C Huang 1, E A Wood 1, M M Cox 1
PMCID: PMC179511  PMID: 9324255

Abstract

We have created a system that utilizes the FLP recombinase of yeast to introduce exogenous cloned DNA reversibly at defined locations in the Escherichia coli chromosome. Recombination target (FRT) sites can be introduced permanently at random locations in the chromosome on a modified Tn5 transposon, now designed so that the inserted FRT can be detected and its location mapped with base pair resolution. FLP recombinase is provided as needed through the regulated expression of its gene on a plasmid. Exogenous DNA is introduced on a cloning vector that contains an FRT, selectable markers, and a replication origin designed to be deleted prior to electroporation for targeting purposes. High yields of targeted integrants are obtained, even in a recA background. This system permits rapid and precise excision of the introduced DNA when needed, without destroying the cells. The efficiency of targeting appears to be affected only modestly by transcription initiation upstream of the chromosomal FRT site. With rare exceptions, FRTs introduced to the bacterial chromosome are targeted with high efficiency regardless of their location. The system should facilitate studies of bacterial genome structure and function, simplify a wide range of chromosomal cloning applications, and generally enhance the utility of E. coli as an experimental organism in biotechnology.

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Selected References

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

  1. Ayres E. K., Thomson V. J., Merino G., Balderes D., Figurski D. H. Precise deletions in large bacterial genomes by vector-mediated excision (VEX). The trfA gene of promiscuous plasmid RK2 is essential for replication in several gram-negative hosts. J Mol Biol. 1993 Mar 5;230(1):174–185. doi: 10.1006/jmbi.1993.1134. [DOI] [PubMed] [Google Scholar]
  2. Baubonis W., Sauer B. Genomic targeting with purified Cre recombinase. Nucleic Acids Res. 1993 May 11;21(9):2025–2029. doi: 10.1093/nar/21.9.2025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bayley C. C., Morgan M., Dale E. C., Ow D. W. Exchange of gene activity in transgenic plants catalyzed by the Cre-lox site-specific recombination system. Plant Mol Biol. 1992 Jan;18(2):353–361. doi: 10.1007/BF00034962. [DOI] [PubMed] [Google Scholar]
  4. Bernet A., Sabatier S., Picketts D. J., Ouazana R., Morlé F., Higgs D. R., Godet J. Targeted inactivation of the major positive regulatory element (HS-40) of the human alpha-globin gene locus. Blood. 1995 Aug 1;86(3):1202–1211. [PubMed] [Google Scholar]
  5. Cherepanov P. P., Wackernagel W. Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene. 1995 May 26;158(1):9–14. doi: 10.1016/0378-1119(95)00193-a. [DOI] [PubMed] [Google Scholar]
  6. Cox M. M. The FLP protein of the yeast 2-microns plasmid: expression of a eukaryotic genetic recombination system in Escherichia coli. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4223–4227. doi: 10.1073/pnas.80.14.4223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Craig N. L. The mechanism of conservative site-specific recombination. Annu Rev Genet. 1988;22:77–105. doi: 10.1146/annurev.ge.22.120188.000453. [DOI] [PubMed] [Google Scholar]
  8. DiSanto J. P., Müller W., Guy-Grand D., Fischer A., Rajewsky K. Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor gamma chain. Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):377–381. doi: 10.1073/pnas.92.2.377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fiering S., Epner E., Robinson K., Zhuang Y., Telling A., Hu M., Martin D. I., Enver T., Ley T. J., Groudine M. Targeted deletion of 5'HS2 of the murine beta-globin LCR reveals that it is not essential for proper regulation of the beta-globin locus. Genes Dev. 1995 Sep 15;9(18):2203–2213. doi: 10.1101/gad.9.18.2203. [DOI] [PubMed] [Google Scholar]
  10. Fiering S., Kim C. G., Epner E. M., Groudine M. An "in-out" strategy using gene targeting and FLP recombinase for the functional dissection of complex DNA regulatory elements: analysis of the beta-globin locus control region. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8469–8473. doi: 10.1073/pnas.90.18.8469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fukushige S., Sauer B. Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7905–7909. doi: 10.1073/pnas.89.17.7905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fuse T., Kodama H., Hayashida N., Shinozaki K., Nishimura M., Iba K. A novel Ti-plasmid-convertible lambda phage vector system suitable for gene isolation by genetic complementation of Arabidopsis thaliana mutants. Plant J. 1995 May;7(5):849–856. doi: 10.1046/j.1365-313x.1995.07050849.x. [DOI] [PubMed] [Google Scholar]
  13. Gage P. J., Sauer B., Levine M., Glorioso J. C. A cell-free recombination system for site-specific integration of multigenic shuttle plasmids into the herpes simplex virus type 1 genome. J Virol. 1992 Sep;66(9):5509–5515. doi: 10.1128/jvi.66.9.5509-5515.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Golic K. G., Lindquist S. The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell. 1989 Nov 3;59(3):499–509. doi: 10.1016/0092-8674(89)90033-0. [DOI] [PubMed] [Google Scholar]
  15. Golic K. G. Local transposition of P elements in Drosophila melanogaster and recombination between duplicated elements using a site-specific recombinase. Genetics. 1994 Jun;137(2):551–563. doi: 10.1093/genetics/137.2.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gu H., Zou Y. R., Rajewsky K. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell. 1993 Jun 18;73(6):1155–1164. doi: 10.1016/0092-8674(93)90644-6. [DOI] [PubMed] [Google Scholar]
  17. Guyer M. S., Reed R. R., Steitz J. A., Low K. B. Identification of a sex-factor-affinity site in E. coli as gamma delta. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):135–140. doi: 10.1101/sqb.1981.045.01.022. [DOI] [PubMed] [Google Scholar]
  18. Huang L. C., Wood E. A., Cox M. M. A bacterial model system for chromosomal targeting. Nucleic Acids Res. 1991 Feb 11;19(3):443–448. doi: 10.1093/nar/19.3.443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jayaram M. Phosphoryl transfer in Flp recombination: a template for strand transfer mechanisms. Trends Biochem Sci. 1994 Feb;19(2):78–82. doi: 10.1016/0968-0004(94)90039-6. [DOI] [PubMed] [Google Scholar]
  20. Johnson R. C., Yin J. C., Reznikoff W. S. Control of Tn5 transposition in Escherichia coli is mediated by protein from the right repeat. Cell. 1982 Oct;30(3):873–882. doi: 10.1016/0092-8674(82)90292-6. [DOI] [PubMed] [Google Scholar]
  21. Lakso M., Pichel J. G., Gorman J. R., Sauer B., Okamoto Y., Lee E., Alt F. W., Westphal H. Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5860–5865. doi: 10.1073/pnas.93.12.5860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lakso M., Sauer B., Mosinger B., Jr, Lee E. J., Manning R. W., Yu S. H., Mulder K. L., Westphal H. Targeted oncogene activation by site-specific recombination in transgenic mice. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6232–6236. doi: 10.1073/pnas.89.14.6232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Leslie N. R., Sherratt D. J. Site-specific recombination in the replication terminus region of Escherichia coli: functional replacement of dif. EMBO J. 1995 Apr 3;14(7):1561–1570. doi: 10.1002/j.1460-2075.1995.tb07142.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Medberry S. L., Dale E., Qin M., Ow D. W. Intra-chromosomal rearrangements generated by Cre-lox site-specific recombination. Nucleic Acids Res. 1995 Feb 11;23(3):485–490. doi: 10.1093/nar/23.3.485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Morris A. C., Schaub T. L., James A. A. FLP-mediated recombination in the vector mosquito, Aedes aegypti. Nucleic Acids Res. 1991 Nov 11;19(21):5895–5900. doi: 10.1093/nar/19.21.5895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. O'Gorman S., Fox D. T., Wahl G. M. Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science. 1991 Mar 15;251(4999):1351–1355. doi: 10.1126/science.1900642. [DOI] [PubMed] [Google Scholar]
  27. Osborne B. I., Wirtz U., Baker B. A system for insertional mutagenesis and chromosomal rearrangement using the Ds transposon and Cre-lox. Plant J. 1995 Apr;7(4):687–701. doi: 10.1046/j.1365-313x.1995.7040687.x. [DOI] [PubMed] [Google Scholar]
  28. Pósfai G., Koob M., Hradecná Z., Hasan N., Filutowicz M., Szybalski W. In vivo excision and amplification of large segments of the Escherichia coli genome. Nucleic Acids Res. 1994 Jun 25;22(12):2392–2398. doi: 10.1093/nar/22.12.2392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Qin M., Lee E., Zankel T., Ow D. W. Site-specific cleavage of chromosomes in vitro through Cre-lox recombination. Nucleic Acids Res. 1995 Jun 11;23(11):1923–1927. doi: 10.1093/nar/23.11.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ramírez-Solis R., Liu P., Bradley A. Chromosome engineering in mice. Nature. 1995 Dec 14;378(6558):720–724. doi: 10.1038/378720a0. [DOI] [PubMed] [Google Scholar]
  31. Sadowski P. D. The Flp recombinase of the 2-microns plasmid of Saccharomyces cerevisiae. Prog Nucleic Acid Res Mol Biol. 1995;51:53–91. [PubMed] [Google Scholar]
  32. Sauer B. Identification of cryptic lox sites in the yeast genome by selection for Cre-mediated chromosome translocations that confer multiple drug resistance. J Mol Biol. 1992 Feb 20;223(4):911–928. doi: 10.1016/0022-2836(92)90252-f. [DOI] [PubMed] [Google Scholar]
  33. Sauer B. Manipulation of transgenes by site-specific recombination: use of Cre recombinase. Methods Enzymol. 1993;225:890–900. doi: 10.1016/0076-6879(93)25056-8. [DOI] [PubMed] [Google Scholar]
  34. Sauer B. Recycling selectable markers in yeast. Biotechniques. 1994 Jun;16(6):1086–1088. [PubMed] [Google Scholar]
  35. Sauer B. Site-specific recombination: developments and applications. Curr Opin Biotechnol. 1994 Oct;5(5):521–527. doi: 10.1016/0958-1669(94)90068-x. [DOI] [PubMed] [Google Scholar]
  36. Senecoff J. F., Bruckner R. C., Cox M. M. The FLP recombinase of the yeast 2-micron plasmid: characterization of its recombination site. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7270–7274. doi: 10.1073/pnas.82.21.7270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Slater S., Maurer R. Simple phagemid-based system for generating allele replacements in Escherichia coli. J Bacteriol. 1993 Jul;175(13):4260–4262. doi: 10.1128/jb.175.13.4260-4262.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Smith A. J., De Sousa M. A., Kwabi-Addo B., Heppell-Parton A., Impey H., Rabbitts P. A site-directed chromosomal translocation induced in embryonic stem cells by Cre-loxP recombination. Nat Genet. 1995 Apr;9(4):376–385. doi: 10.1038/ng0495-376. [DOI] [PubMed] [Google Scholar]
  39. Van Deursen J., Fornerod M., Van Rees B., Grosveld G. Cre-mediated site-specific translocation between nonhomologous mouse chromosomes. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7376–7380. doi: 10.1073/pnas.92.16.7376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Waite L. L., Cox M. M. A protein dissociation step limits turnover in FLP recombinase-mediated site-specific recombination. J Biol Chem. 1995 Oct 6;270(40):23409–23414. doi: 10.1074/jbc.270.40.23409. [DOI] [PubMed] [Google Scholar]
  41. Wang P., Anton M., Graham F. L., Bacchetti S. High frequency recombination between loxP sites in human chromosomes mediated by an adenovirus vector expressing Cre recombinase. Somat Cell Mol Genet. 1995 Nov;21(6):429–441. doi: 10.1007/BF02310209. [DOI] [PubMed] [Google Scholar]
  42. Weinreich M. D., Yigit H., Reznikoff W. S. Overexpression of the Tn5 transposase in Escherichia coli results in filamentation, aberrant nucleoid segregation, and cell death: analysis of E. coli and transposase suppressor mutations. J Bacteriol. 1994 Sep;176(17):5494–5504. doi: 10.1128/jb.176.17.5494-5504.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wu H. Y., Shyy S. H., Wang J. C., Liu L. F. Transcription generates positively and negatively supercoiled domains in the template. Cell. 1988 May 6;53(3):433–440. doi: 10.1016/0092-8674(88)90163-8. [DOI] [PubMed] [Google Scholar]
  44. Xu T., Rubin G. M. Analysis of genetic mosaics in developing and adult Drosophila tissues. Development. 1993 Apr;117(4):1223–1237. doi: 10.1242/dev.117.4.1223. [DOI] [PubMed] [Google Scholar]
  45. Yu G. L., Blackburn E. H. Amplification of tandemly repeated origin control sequences confers a replication advantage on rDNA replicons in Tetrahymena thermophila. Mol Cell Biol. 1990 May;10(5):2070–2080. doi: 10.1128/mcb.10.5.2070. [DOI] [PMC free article] [PubMed] [Google Scholar]

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