Transgenic Research

, Volume 16, Issue 4, pp 405–413 | Cite as

Adopting the good reFLEXes when generating conditional alterations in the mouse genome

Perspective

Abstract

Major advances have been made in the use of the Cre/loxP system for conditional gene targeting in the mouse. By combining the ability of Cre recombinase to invert or excise a DNA fragment, depending upon the orientation of the flanking loxP sites, and the use of wild-type loxP and variant lox511 sites, we devised an efficient and reliable Cre-mediated genetic switch, called FLEX, through which expression of a given gene can be turned off, while expression of another one can be simultaneously turned on. We discuss how this innovative, flexible and powerful approach, which virtually adapts to any kind of site-specific recombinase (e.g., Cre and Flp recombinases), can be used to easily generate, even at high throughput and genome wide scale, many genetic modifications in a conditional manner, including those which were considered as difficult or impossible to achieve.

Keywords

FRT Heterotypic sites Conditional allele Recombinase-mediated cassette exchange (RMCE) Point mutation Gene trap 

References

  1. Abremski K, Hoess R, Sternberg N (1983) Studies on the properties of P1 site-specific recombination: evidence for topologically unlinked products following recombination. Cell 32:1301–1311PubMedCrossRefGoogle Scholar
  2. Araki K, Araki M, Yamamura K (1997) Targeted integration of DNA using mutant lox sites in embryonic stem cells. Nucleic Acids Res 25:868–872PubMedCrossRefGoogle Scholar
  3. Araki K, Imaizumi T, Sekimoto T, Yoshinobu K, Yoshimuta J, Akizuki M, Miura K, Araki M, Yamamura K (1999) Exchangeable gene trap using the Cre/mutated lox system. Cell Mol Biol 45:737–750PubMedGoogle Scholar
  4. Araki K, Araki M, Yamamura K (2002) Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites. Nucleic Acids Res 30:e103PubMedCrossRefGoogle Scholar
  5. Austin CP, Battey JF, Bradley A, Bucan M, Capecchi M, Collins FS, Dove WF, Duyk G, Dymecki S, Eppig JT, Grieder FB, Heintz N, Hicks G, Insel TR, Joyner A, Koller BH, Lloyd KC, Magnuson T, Moore MW, Nagy A, Pollock JD, Roses AD, Sands AT, Seed B, Skarnes WC, Snoddy J, Soriano P, Stewart DJ, Stewart F, Stillman B, Varmus H, Varticovski L, Verma IM, Vogt TF, von Melchner H, Witkowski J, Woychik RP, Wurst W, Yancopoulos GD, Young SG, Zambrowicz B (2004) The knockout mouse project. Nat Genet 36:921–924PubMedCrossRefGoogle Scholar
  6. Auwerx J, Avner P, Baldock R, Ballabio A, Balling R, Barbacid M, Berns A, Bradley A, Brown S, Carmeliet P, Chambon P, Cox R, Davidson D, Davies K, Duboule D, Forejt J, Granucci F, Hastie N, de Angelis MH, Jackson I, Kioussis D, Kollias G, Lathrop M, Lendahl U, Malumbres M, von Melchner H, Muller W, Partanen J, Ricciardi-Castagnoli P, Rigby P, Rosen B, Rosenthal N, Skarnes B, Stewart AF, Thornton J, Tocchini-Valentini G, Wagner E, Wahli W, Wurst W (2004) The European dimension for the mouse genome mutagenesis program. Nat Genet 36:925–927PubMedCrossRefGoogle Scholar
  7. Brakebusch C, Grose R, Quondamatteo F, Ramirez A, Jorcano JL, Pirro A, Svensson M, Herken R, Sasaki T, Timpl R, Werner S, Fassler R (2000) Skin and hair follicle integrity is crucially dependent on beta 1 integrin expression on keratinocytes. EMBO J 19:3990–4003PubMedCrossRefGoogle Scholar
  8. Branda CS, Dymecki SM (2004) Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Dev Cell 6:7–28PubMedCrossRefGoogle Scholar
  9. Brocard J, Warot X, Wendling O, Messaddeq N, Vonesch JL, Chambon P, Metzger D (1997) Spatio-temporally controlled site-specific somatic mutagenesis in the mouse. Proc Natl Acad Sci U S A 94:14559–14563PubMedCrossRefGoogle Scholar
  10. Buchholz F, Ringrose L, Angrand PO, Rossi F, Stewart AF (1996) Different thermostabilities of FLP and Cre recombinases: implications for applied site-specific recombination. Nucleic Acids Res 24:4256–4262PubMedCrossRefGoogle Scholar
  11. Capecchi MR (1989) Altering the genome by homologous recombination. Science 244:1288–1292PubMedCrossRefGoogle Scholar
  12. Feng YQ, Seibler J, Alami R, Eisen A, Westerman KA, Leboulch P, Fiering S, Bouhassira EE (1999) Site-specific chromosomal integration in mammalian cells: highly efficient Cre recombinase-mediated cassette exchange. J Mol Biol 292:779–785PubMedCrossRefGoogle Scholar
  13. Forster A, Pannell R, Drynan LF, Codrington R, Daser A, Metzler M, Lobato MN, Rabbitts TH (2005) The invertor knock-in conditional chromosomal translocation mimic. Nat Methods 2:27–30PubMedCrossRefGoogle Scholar
  14. Garcia EL, Mills AA (2002) Getting around lethality with Cre-mediated excision. Semin Cell Dev Biol 13:151–158PubMedCrossRefGoogle Scholar
  15. Guenet JL (2005) The mouse genome. Genome Res 15:1729–1740PubMedCrossRefGoogle Scholar
  16. Hansen J, Floss T, Van Sloun P, Füchtbauer EM, Vauti F, Arnold HH, Schnütgen F, Wurst W, von Melchner H, Ruiz P (2003) A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome. Proc Natl Acad Sci U S A 100:9918–9922PubMedCrossRefGoogle Scholar
  17. Kano M, Igarashi H, Saito I, Masuda M (1998) Cre-loxP-mediated DNA flip-flop in mammalian cells leading to alternate expression of retrovirally transduced genes. Biochem Biophys Res Commun 248:806–811PubMedCrossRefGoogle Scholar
  18. Kellendonk C, Tronche F, Casanova E, Anlag K, Opherk C, Schutz G (1999) Inducible site-specific recombination in the brain. J Mol Biol 285:175–182PubMedCrossRefGoogle Scholar
  19. Kolb AF (2001) Selection-marker-free modification of the murine β-casein gene using a lox2272 site. Anal Biochem 290:260–271PubMedCrossRefGoogle Scholar
  20. Kulessa H, Hogan BL (2002) Generation of a loxP flanked bmp4loxP–lacZ allele marked by conditional lacZ expression. Genesis 32:66–68PubMedCrossRefGoogle Scholar
  21. Lam KP, Rajewsky K (1998) Rapid elimination of autoreactive B cells demonstrated by Cre-induced change in B cell antigen receptor specificity in vivo. Proc Natl Acad Sci U S A 95:13171–13175PubMedCrossRefGoogle Scholar
  22. Lauth M, Moerl K, Barski JJ, Meyer M (2000) Characterization of Cre-mediated cassette exchange after plasmid microinjection in fertilized mouse oocytes. Genesis 27:153–158PubMedCrossRefGoogle Scholar
  23. Lee G, Saito I (1998) Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene 216:55–65PubMedCrossRefGoogle Scholar
  24. Lobe CG, Koop KE, Kreppner W, Lomeli H, Gertenstein M, Nagy A (1999) Dev Biol 208:281–292PubMedCrossRefGoogle Scholar
  25. Luche H, Weber O, Nageswara Rao T, Blum C, Fehling HJ (2007) Faithful activation of an extra-bright red fluorescent protein in “knock-in” Cre-reporter mice ideally suited for lineage tracing studies. Eur J Immunol 37:43–53PubMedCrossRefGoogle Scholar
  26. Martin DI, Whitelaw E (1996) The vagaries of variegating transgenes. Bioessays 18:919–923PubMedCrossRefGoogle Scholar
  27. Metzger D, Chambon P (2001) Site- and time-specific gene targeting in the mouse. Methods 24:71–80PubMedCrossRefGoogle Scholar
  28. Mlynarova L, Libantova J, Vrba L, Nap JP (2002) The promiscuity of heterospecific lox sites increases dramatically in the presence of palindromic DNA. Gene 296:129–137PubMedCrossRefGoogle Scholar
  29. Montoliu L, Chavez S, Vidal M (2000) Variegation associated with lacZ in transgenic animals: a warning note. Transgenic Res 9:237–239PubMedCrossRefGoogle Scholar
  30. Moon AM, Capecchi MR (2000) Fgf8 is required for outgrowth and patterning of the limbs. Nat Genet 26:455–459PubMedCrossRefGoogle Scholar
  31. Müller U (1999) Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis. Mech Dev 82:3–21PubMedCrossRefGoogle Scholar
  32. Nagy A (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis 26:99–109PubMedCrossRefGoogle Scholar
  33. Nord AS, Chang PJ, Conklin BR, Cox AV, Harper CA, Hicks GG, Huang CC, Johns SJ, Kawamoto M, Liu S, Meng EC, Morris JH, Rossant J, Ruiz P, Skarnes WC, Soriano P, Stanford WL, Stryke D, von Melchner H, Wurst W, Yamamura K, Young SG, Babbitt PC, Ferrin TE (2006) The International Gene Trap Consortium Website: a portal to all publicly available gene trap cell lines in mouse. Nucleic Acids Res 34:D642–D648PubMedCrossRefGoogle Scholar
  34. Novak A, Guo C, Yang W, Nagy A, Lobe CG (2000) Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 28:147–155PubMedCrossRefGoogle Scholar
  35. Oberdoerffer P, Otipoby KL, Maruyama M, Rajewski K (2003) Unidirectional Cre-mediated genetic inversion in mice using the mutant loxP pair lox66/lox71. Nucleic Acids Res 31:e140PubMedCrossRefGoogle Scholar
  36. O’Gorman S, Fox DT, Wahl GM (1991) Recombinase mediated gene activation and site-specific integration in mammalian cells. Science 251:1351–1355PubMedCrossRefGoogle Scholar
  37. Qu S, Rinehart C, Wu HH, Wang SE, Carter B, Xin H, Kotlikoff M, Arteaga CL (2006) Gene targeting of ErbB3 using a Cre-mediated unidirectional DNA inversion strategy. Genesis 44:477–486PubMedCrossRefGoogle Scholar
  38. Ringrose L, Chabanis S, Angrand PO, Woodroofe C, Stewart AF (1999) Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA FLEXibility at short distances. EMBO J 18:6630–6641PubMedCrossRefGoogle Scholar
  39. Roberts CW, Leroux MM, Fleming MD, Orkin SH (2002) Highly penetrant, rapid tumorigenesis through conditional inversion of the tumor suppressor gene Snf5. Cancer Cell 2:415–425PubMedCrossRefGoogle Scholar
  40. Samokhvalov IM, Thomson AM, Lalancette C, Liakhovitskaia A, Ure J, Medvinsky A (2006) Multifunctional reversible knockout/reporter system enabling fully functional reconstitution of the AML1/Runx1 locus and rescue of hematopoiesis. Genesis 44:115–121PubMedCrossRefGoogle Scholar
  41. Sauer B, Henderson N (1988) Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A 85:5166–5170PubMedCrossRefGoogle Scholar
  42. Sauer B (1998) Inducible Gene targeting in mice using the Cre/lox system. Methods 14:381–392PubMedCrossRefGoogle Scholar
  43. Schnütgen F, Doerflinger N, Calleja C, Wendling O, Chambon P, Ghyselinck NB (2003) A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Nat Biotechnol 21:562–565PubMedCrossRefGoogle Scholar
  44. Schnütgen F, De-Zolt S, Van Sloun P, Hollatz M, Floss T, Hansen J, Altschmied J, Seisenberger C, Ghyselinck NB, Ruiz P, Chambon P, Wurst W, von Melchner H (2005) Genomewide production of multipurpose alleles for the functional analysis of the mouse genome. Proc Natl Acad Sci U S A 102:7221–7226PubMedCrossRefGoogle Scholar
  45. Schwenk F, Sauer B, Kukoc N, Hoess R, Müller W, Kocks C, Kühn R, Rajewsky K (1997) Generation of Cre recombinase-specific monoclonal antibodies, able to characterize the pattern of Cre expression in cre-transgenic mouse strains. J Immunol Methods 207:203–212PubMedCrossRefGoogle Scholar
  46. Siegel RW, Jain R, Bradbury A (2001) Using an in vivo phagemid system to identify non-compatible loxP sequences. FEBS Lett 499:147–153PubMedCrossRefGoogle Scholar
  47. Shmerling D, Danzer CP, Mao X, Boisclair J, Haffner M, Lemaistre M, Schuler V, Kaeslin E, Korn R, Burki K, Ledermann B, Kinzel B, Muller M (2005) Strong and ubiquitous expression of transgenes targeted into the beta-actin locus by Cre/lox cassette replacement. Genesis 42:229–235PubMedCrossRefGoogle Scholar
  48. Skvorak K, Vissel B, Homanics GE (2006) Production of conditional point mutant knockin mice. Genesis 44:345–353PubMedCrossRefGoogle Scholar
  49. Theis M, Mas C, Doring B, Kruger O, Herrera P, Meda P, Willecke K (2001) General and conditional replacement of connexin43-coding DNA by a lacZ reporter gene for cell-autonomous analysis of expression. Cell Commun Adhes 8:383–386PubMedCrossRefGoogle Scholar
  50. Ungrin MD, Harrington L (2006) Strict control of telomerase activation using Cre-mediated inversion. BMC Biotechnol 6:10PubMedCrossRefGoogle Scholar
  51. Vooijs M, Jonkers J, Berns A (2001) A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent. EMBO Rep 2:292–297PubMedCrossRefGoogle Scholar
  52. Wiles MV, Vauti F, Otte J, Füchtbauer EM, Ruiz P, Füchtbauer A, Arnold HH, Lehrach H, Metz T, von Melchner H, Wurst W (2000) Establishment of a gene-trap sequence tag library to generate mutant mice from embryonic stem cells. Nat Genet 24:13–14PubMedCrossRefGoogle Scholar
  53. Xin HB, Deng KY, Shui B, Qu S, Sun Q, Lee J, Greene KS, Wilson J, Yu Y, Feldman M, Kotlikoff MI (2005) Gene trap and gene inversion methods for conditional gene inactivation in the mouse. Nucleic Acids Res 33:e14PubMedCrossRefGoogle Scholar
  54. Zhang Z, Lutz B (2002) Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein. Nucleic Acids Res 30:e90PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  1. 1.Department of Molecular HaematologyUniversity of Frankfurt Medical SchoolFrankfurt am MainGermany
  2. 2.IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Inserm U596, CNRS UMR7104IllkirchFrance
  3. 3.Université Louis PasteurStrasbourgFrance
  4. 4.IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire)Illkirch CedexFrance

Personalised recommendations