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Transgenic Research

, Volume 19, Issue 3, pp 363–371 | Cite as

Zinc-finger nucleases: a powerful tool for genetic engineering of animals

  • Séverine Rémy
  • Laurent Tesson
  • Séverine Ménoret
  • Claire Usal
  • Andrew M. Scharenberg
  • Ignacio AnegonEmail author
Review

Abstract

The generation of genetically modified animals or plants with gene-targeted deletions or modifications is a powerful tool to analyze gene function, study disease and produce organisms of economical interest. Until recently, the generation of animals with gene targeted manipulations has been accomplished by homologous recombination (HR) in embryonic stem (ES) cells or cloning through nuclear transfer and has been limited to a few species. Recently, a new technology based on the use of gene-targeted zinc-finger nucleases (ZFNs) was developed and used for the generation of organisms with gene-targeted deletions and/or modifications when combined with HR. ZFNs have been used to generate modified organisms such as plants, Drosophila, zebra fish and rats with gene-targeted mutations. This perspective manuscript is a short review on the use of ZFNs for the genetic engineering of plants and animals, with particular emphasis on our recent work involving rats. We also discuss the application of other targeted nucleases, including homing endonucleases. Microinjection of plasmid or mRNA for ZFNs into rat embryos allowed targeted, rapid, complete, permanent and heritable disruption of endogenous loci. The application of ZFNs to generate gene-targeted knockouts in species where ES cells or cloning techniques are not available is an important new development to answer fundamental biological questions and develop models of economical interest such as for the production of humanized antibodies. Further refinements of ZFN technology in combination with HR may allow knock-ins in early embryos even in species where ES cells or cloning techniques are available.

Keywords

Zinc finger nucleases Targeted transgenesis Rat Gene knockout Homologous recombination Animal models 

Notes

Acknowledgments

This work was in part funded by the Région Pays de la Loire through Biogenouest and IMBIO programs as well as by the IBiSA program, and NIH grant UL1DE019582 to AMS.

References

  1. Aitman TJ, Critser JK, Cuppen E, Dominiczak A, Fernandez-Suarez XM, Flint J, Gauguier D, Geurts AM, Gould M, Harris PC, Holmdahl R, Hubner N, Izsvak Z, Jacob HJ, Kuramoto T, Kwitek AE, Marrone A, Mashimo T, Moreno C, Mullins J, Mullins L, Olsson T, Pravenec M, Riley L, Saar K, Serikawa T, Shull JD, Szpirer C, Twigger SN, Voigt B, Worley K (2008) Progress and prospects in rat genetics: a community view. Nat Genet 40(5):516–522CrossRefPubMedGoogle Scholar
  2. Arnould S, Chames P, Perez C, Lacroix E, Duclert A, Epinat JC, Stricher F, Petit AS, Patin A, Guillier S, Rolland S, Prieto J, Blanco FJ, Bravo J, Montoya G, Serrano L, Duchateau P, Paques F (2006) Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets. J Mol Biol 355(3):443–458CrossRefPubMedGoogle Scholar
  3. Ashworth J, Havranek JJ, Duarte CM, Sussman D, Monnat RJ Jr, Stoddard BL, Baker D (2006) Computational redesign of endonuclease DNA binding and cleavage specificity. Nature 441(7093):656–659CrossRefPubMedGoogle Scholar
  4. Barnes DE, Stamp G, Rosewell I, Denzel A, Lindahl T (1998) Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice. Curr Biol 8(25):1395–1398CrossRefPubMedGoogle Scholar
  5. Batista LF, Kaina B, Meneghini R, Menck CF (2009) How DNA lesions are turned into powerful killing structures: insights from UV-induced apoptosis. Mutat Res 681(2–3):197–208PubMedGoogle Scholar
  6. Beerli RR, Barbas CF III (2002) Engineering polydactyl zinc-finger transcription factors. Nat Biotechnol 20(2):135–141CrossRefPubMedGoogle Scholar
  7. Beumer KJ, Trautman JK, Bozas A, Liu J-L, Rutter J, Gall JG, Carroll D (2008) Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Proc Natl Acad Sci USA 105(50):19821–19826CrossRefPubMedGoogle Scholar
  8. Bibikova M, Golic M, Golic KG, Carroll D (2002) Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161(3):1169–1175PubMedGoogle Scholar
  9. Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300(5620):764CrossRefPubMedGoogle Scholar
  10. Bozas A, Beumer KJ, Trautman JK, Carroll D (2009) Genetic analysis of zinc-finger nuclease-induced gene targeting in Drosophila. Genetics 182(3):641–651Google Scholar
  11. Bronson SK, Plaehn EG, Kluckman KD, Hagaman JR, Maeda N, Smithies O (1996) Single-copy transgenic mice with chosen-site integration. Proc Natl Acad Sci USA 93(17):9067–9072CrossRefPubMedGoogle Scholar
  12. Buehr M, Meek S, Blair K, Yang J, Ure J, Silva J, McLay R, Hall J, Ying Q, Smith A (2008) Capture of authentic embryonic stem cells from rat blastocysts. Cell 135(7):1287–1298CrossRefPubMedGoogle Scholar
  13. Cai C, Doyon Y, Ainley W, Miller J, DeKelver R, Moehle E, Rock J, Lee Y-L, Garrison R, Schulenberg L, Blue R, Worden A, Baker L, Faraji F, Zhang L, Holmes M, Rebar E, Collingwood T, Rubin-Wilson B, Gregory P, Urnov F, Petolino J (2009) Targeted transgene integration in plant cells using designed zinc finger nucleases. Plant Mol Biol 69(6):699CrossRefPubMedGoogle Scholar
  14. Cannata F, Brunet E, Perrouault L, Roig V, Ait-Si-Ali S, Asseline U, Concordet JP, Giovannangeli C (2008) Triplex-forming oligonucleotide-orthophenanthroline conjugates for efficient targeted genome modification. Proc Natl Acad Sci USA 105(28):9576–9581CrossRefPubMedGoogle Scholar
  15. Carroll D (2008) Progress and prospects: zinc-finger nucleases as gene therapy agents. Gene Ther 15(22):1463–1468CrossRefPubMedGoogle Scholar
  16. Carroll D, Beumer KJ, Morton JJ, Bozas A, Trautman JK (2008) Gene targeting in Drosophila and Caenorhabditis elegans with zinc-finger nucleases. Methods Mol Biol 435:63–77CrossRefPubMedGoogle Scholar
  17. Charreau B, Tesson L, Soulillou JP, Pourcel C, Anegon I (1996) Transgenic rats: technical aspects and models. Transgenic Res 5:223–234CrossRefPubMedGoogle Scholar
  18. Cozzi J, Wan E, Jacquet C, Fraichard A, Cherifi Y, Zhou Q (2009) Procedures for somatic cell nuclear transfer in the rat. Methods in mol biol “rat genomics: gene identification, functional genomics and model applications”, vol 561, pp 73–88Google Scholar
  19. Danilova N, Bussmann J, Jekosch K, Steiner LA (2005) The immunoglobulin heavy-chain locus in zebrafish: identification and expression of a previously unknown isotype, immunoglobulin Z. Nat Immunol 6(3):295–302CrossRefPubMedGoogle Scholar
  20. Derijck A, van der Heijden G, Giele M, Philippens M, de Boer P (2008) DNA double-strand break repair in parental chromatin of mouse zygotes, the first cell cycle as an origin of de novo mutation. Hum Mol Genet 17(13):1922–1937CrossRefPubMedGoogle Scholar
  21. Donigan KA, Sweasy JB (2009) Sequence context-specific mutagenesis and base excision repair. Mol Carcinog 48(4):362–368CrossRefPubMedGoogle Scholar
  22. Doyon Y, McCammon JM, Miller JC, Faraji F, Ngo C, Katibah GE, Amora R, Hocking TD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Amacher SL (2008) Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol 26(6):702CrossRefPubMedGoogle Scholar
  23. Foley JE, Yeh J-RJ, Maeder ML, Reyon D, Sander JD, Peterson RT, Joung JK (2009) Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by oligomerized pool engineering (OPEN). PLoS ONE 4(2):e4348CrossRefPubMedGoogle Scholar
  24. Forand A, Fouchet P, Lahaye JB, Chicheportiche A, Habert R, Bernardino-Sgherri J (2009) Similarities and differences in the in vivo response of mouse neonatal gonocytes and spermatogonia to genotoxic stress. Biol Reprod 80(5):860–873CrossRefPubMedGoogle Scholar
  25. Geurts AM, Cost C, Rémy S, Cui X, Tesson L, Usal C, Menoret S, Jacob H, Anegon I, Buelow R (2009a) Generation of gene-specific mutated rats using zinc finger nucleases. Methods in mol biol rat genomics: gene identification, functional genomics and model applications. Humana Press (in press)Google Scholar
  26. Geurts AM, Cost GJ, Miller JC, Freyvert Y, Zeitler B, Choi VM, Jenkins SS, Wood A, Cui X, Meng X, Vincent A, Lam S, DeKelver RC, Michalkiewicz M, Schilling R, Foeckler J, Kalloway S, Weiler H, Ménoret S, Anegon I, Davis GD, Sullivan P, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jacob HJ, Buelow R (2009b) Knockout rats produced via embryo pronuclear microinjection of designed zinc finger nucleases. Science 325(5939):433CrossRefPubMedGoogle Scholar
  27. Grizot S, Smith J, Daboussi F, Prieto J, Redondo P, Merino N, Villate M, Thomas S, Lemaire L, Montoya G, Blanco FJ, Paques F, Duchateau P (2009) Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease. Nucleic Acids Res. doi: 10.1093/nar/gkp548
  28. Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, DeKelver RC, Katibah GE, Amora R, Boydston EA, Zeitler B, Meng X, Miller JC, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R (2009) Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol 27(9):851–857Google Scholar
  29. Isalan M, Klug A, Choo Y (2001) A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter. Nat Biotechnol 19(7):656–660CrossRefPubMedGoogle Scholar
  30. Jacob HJ (2009) The rat: a model used in biomedical research. Methods in mol biol “rat genomics: gene identification, functional genomics and model applications”. Humana Press (in press)Google Scholar
  31. Kandavelou K, Chandrasegaran S (2009) Custom-designed molecular scissors for site-specific manipulation of the plant and Mammalian genomes. Methods Mol Biol 544:617–636CrossRefPubMedGoogle Scholar
  32. Kawamata M, Ochiya T (2009) Establishment of embryonic stem cells from rat blastocysts. Methods in mol biol “rat genomics: gene identification, functional genomics and model applications”. Humana Press (in press)Google Scholar
  33. Kim YG, Shi Y, Berg JM, Chandrasegaran S (1997) Site-specific cleavage of DNA-RNA hybrids by zinc finger/FokI cleavage domain fusions. Gene 203(1):43–49CrossRefPubMedGoogle Scholar
  34. Kitada K, Ishishita S, Tosaka K, Takahashi R, Ueda M, Keng VW, Horie K, Takeda J (2007) Transposon-tagged mutagenesis in the rat. Nat Methods 4(2):131–133CrossRefPubMedGoogle Scholar
  35. Kitamura D, Roes J, Kuhn R, Rajewsky K (1991) A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature 350(6317):423–426CrossRefPubMedGoogle Scholar
  36. Li P, Tong C, Mehrian-Shai R, Jia L, Wu N, Yan Y, Maxson RE, Schulze EN, Song H, Hsieh CL, Pera MF, Ying Q (2008) Germline competent embryonic stem cells derived from rat blastocysts. Cell 135:1299–1310CrossRefPubMedGoogle Scholar
  37. Lo AW, Sprung CN, Fouladi B, Pedram M, Sabatier L, Ricoul M, Reynolds GE, Murnane JP (2002) Chromosome instability as a result of double-strand breaks near telomeres in mouse embryonic stem cells. Mol Cell Biol 22(13):4836–4850CrossRefPubMedGoogle Scholar
  38. Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D (2002) Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295(5556):868–872CrossRefPubMedGoogle Scholar
  39. Lombardo A, Genovese P, Beausejour CM, Colleoni S, Lee YL, Kim KA, Ando D, Urnov FD, Galli C, Gregory PD, Holmes MC, Naldini L (2007) Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol 25(11):1298–1306CrossRefPubMedGoogle Scholar
  40. Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, Jiang T, Foley JE, Winfrey RJ, Townsend JA, Unger-Wallace E, Sander JD, Muller-Lerch F, Fu F, Pearlberg J, Gobel C, Dassie JP, Pruett-Miller SM, Porteus MH, Sgroi DC, Iafrate AJ, Dobbs D, McCray PB Jr, Cathomen T, Voytas DF, Joung JK (2008) Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell 31(2):294–301CrossRefPubMedGoogle Scholar
  41. Mani M, Smith J, Kandavelou K, Berg JM, Chandrasegaran S (2005) Binding of two zinc finger nuclease monomers to two specific sites is required for effective double-strand DNA cleavage. Biochem Biophys Res Commun 334(4):1191–1197CrossRefPubMedGoogle Scholar
  42. Mattei E, Corbi N, Di Certo MG, Strimpakos G, Severini C, Onori A, Desantis A, Libri V, Buontempo S, Floridi A, Fanciulli M, Baban D, Davies KE, Passananti C (2007) Utrophin up-regulation by an artificial transcription factor in transgenic mice. PLoS ONE 2(1):e774CrossRefPubMedGoogle Scholar
  43. Ménoret S, Remy S, Usal C, Tesson L, Anegon I (2009) Generation of transgenic rats by microinjection of short DNA fragments. Methods in mol biol “rat genomics: gene identification, functional genomics and model applications”. Humana Press (in press)Google Scholar
  44. Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA, Gregory PD, Pabo CO, Rebar EJ (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25(7):778–785CrossRefPubMedGoogle Scholar
  45. Moehle EA, Rock JM, Lee YL, Jouvenot Y, DeKelver RC, Gregory PD, Urnov FD, Holmes MC (2007) Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc Natl Acad Sci USA 104(9):3055–3060CrossRefPubMedGoogle Scholar
  46. Monnat R Jr, Scharenberg A, Stoddard B (2008) Progress in engineering homing endonucleases for gene targeting: ten years after structures. In: Bertolotti R, Ozawa K (eds) Progress in gene therapy volume 3: autologous and cancer stem cell gene therapy. World Scientific Publishers, SingaporeGoogle Scholar
  47. Mullins JJ, Peters J, Ganten D (1990) Fulminant hypertension in transgenic rats harbouring the mouse Ren-2 gene. Nature 344:541–544CrossRefPubMedGoogle Scholar
  48. Pabo CO, Peisach E, Grant RA (2001) Design and selection of novel Cys2His2 zinc finger proteins. Annu Rev Biochem 70(1):313–340CrossRefPubMedGoogle Scholar
  49. Pandita TK, Richardson C (2009) Chromatin remodeling finds its place in the DNA double-strand break response. Nucleic Acids Res 37(5):1363–1377CrossRefPubMedGoogle Scholar
  50. Paques F, Duchateau P (2007) Meganucleases and DNA double-strand break-induced recombination: perspectives for gene therapy. Curr Gene Ther 7(1):49–66CrossRefPubMedGoogle Scholar
  51. Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O, Wang N, Lee G, Bartsevich VV, Lee Y-L, Guschin DY, Rupniewski I, Waite AJ, Carpenito C, Carroll RG, Orange JS, Urnov FD, Rebar EJ, Ando D, Gregory PD, Riley JL, Holmes MC, June CH (2008) Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol 26(7):808CrossRefPubMedGoogle Scholar
  52. Pfeifer A (2006) Lentiviral transgenesis—a versatile tool for basic research and gene therapy. Curr Gene Ther 6(4):535–542CrossRefPubMedGoogle Scholar
  53. Phillips ER, McKinnon PJ (2007) DNA double-strand break repair and development. Oncogene 26(56):7799–7808CrossRefPubMedGoogle Scholar
  54. Porteus M (2008). Design and testing of zinc finger nucleases for use in mammalian cells. Methods in Mol Biol. “Chromosomal mutagenesis”, vol 435. Humana Press, pp 47–61Google Scholar
  55. Rémy S, NGuyen T, Ménoret S, Tesson L, Usal C, Anegon I (2009) The use of lentiviral vectors to obtain transgenic rats. Methods in mol biol rat genomics: gene identification, functional genomics and model applications. Humana Press (in press)Google Scholar
  56. Sander JD, Zaback P, Joung JK, Voytas DF, Dobbs D (2007) Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool. Nucleic Acids Res 35((suppl_2)):W599–W605CrossRefPubMedGoogle Scholar
  57. Santiago Y, Chan E, Liu PQ, Orlando S, Zhang L, Urnov FD, Holmes MC, Guschin D, Waite A, Miller JC, Rebar EJ, Gregory PD, Klug A, Collingwood TN (2008) Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc Natl Acad Sci USA 105(15):5809–5814CrossRefPubMedGoogle Scholar
  58. Shinohara T, Kato M, Takehashi M, Lee J, Chuma S, Nakatsuji N, Kanatsu-Shinohara M, Hirabayashi M (2006) Rats produced by interspecies spermatogonial transplantation in mice and in vitro microinsemination. Proc Natl Acad Sci USA 103(37):13624–13628CrossRefPubMedGoogle Scholar
  59. Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Choi VM, Rock JM, Wu Y-Y, Katibah GE, Zhifang G, McCaskill D, Simpson MA, Blakeslee B, Greenwalt SA, Butler HJ, Hinkley SJ, Zhang L, Rebar EJ, Gregory PD, Urnov FD (2009) Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459(7245):437CrossRefPubMedGoogle Scholar
  60. Stoddard BL, Scharenberg AM, Monnat RJ Jr (2008) Advances in engineering homing endonucleases for gene targeting: ten years after structures. In: Bertolotti R, Ozawa K (eds) Chapter 6 in progress in gene therapy 3: autologous and cancer stem cell gene therapy. World Scientific Press, Hackensack, NJ, pp 135–167Google Scholar
  61. Sussman R (2007) DNA repair capacity of zebrafish. Proc Natl Acad Sci USA 104(33):13379–13383CrossRefPubMedGoogle Scholar
  62. Szczepek M, Brondani V, Buchel J, Serrano L, Segal DJ, Cathomen T (2007) Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol 25(7):786–793CrossRefPubMedGoogle Scholar
  63. Takeuchi R, Certo M, Caprara MG, Scharenberg AM, Stoddard BL (2009) Optimization of in vivo activity of a bifunctional homing endonuclease and maturase reverses evolutionary degradation. Nucleic Acids Res 37(3):877–890CrossRefPubMedGoogle Scholar
  64. Tesson L, Cozzi J, Menoret S, Remy S, Usal C, Fraichard A, Anegon I (2005) Transgenic modifications of the rat genome. Transgenic Res 14(5):531–546CrossRefPubMedGoogle Scholar
  65. Townsend JA, Wright DA, Winfrey RJ, Fu F, Maeder ML, Joung JK, Voytas DF (2009) High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature 459(7245):442CrossRefPubMedGoogle Scholar
  66. Ueda S, Kawamata M, Teratani T, Shimizu T, Tamai Y, Ogawa H, Hayashi K, Tsuda H, Ochiya T (2008) Establishment of rat embryonic stem cells and making of chimera rats. PLoS One 3(7):e2800CrossRefPubMedGoogle Scholar
  67. Urnov FD, Miller JC, Lee Y-L, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PD, Holmes MC (2005) Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435(7042):646CrossRefPubMedGoogle Scholar
  68. Volna P, Jarjour J, Baxter S, Roffler SR, Monnat RJ Jr, Stoddard BL, Scharenberg AM (2007) Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases. Nucleic Acids Res 35(8):2748–2758CrossRefPubMedGoogle Scholar
  69. Wright DA, Thibodeau-Beganny S, Sander JD, Winfrey RJ, Hirsh AS, Eichtinger M, Fu F, Porteus MH, Dobbs D, Voytas DF, Joung JK (2006) Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nat Protocols 1(4):1637CrossRefGoogle Scholar
  70. Yang M, Djukanovic V, Stagg J, Lenderts B, Bidney D, Falco SC, Lyznik LA (2009) Targeted mutagenesis in the progeny of maize transgenic plants. Plant Mol Biol 70(6):669–679CrossRefPubMedGoogle Scholar
  71. Zan Y, Haag JD, Chen KS, Shepel LA, Wigington D, Wang YR, Hu R, Lopez-Guajardo CC, Brose HL, Porter KI, Leonard RA, Hitt AA, Schommer SL, Elegbede AF, Gould MN (2003) Production of knockout rats using ENU mutagenesis and a yeast-based screening assay. Nat Biotechnol 21(6):645–651CrossRefPubMedGoogle Scholar
  72. Zhou Q, Renard JP, Le Friec G, Brochard V, Beaujean N, Cherifi Y, Fraichard A, Cozzi J (2003) Generation of fertile cloned rats by regulating oocyte activation. Science 302:1179CrossRefPubMedGoogle Scholar
  73. Zou X, Ayling C, Xian J, Piper TA, Barker PJ, Bruggemann M (2001) Truncation of the mu heavy chain alters BCR signalling and allows recruitment of CD5+ B cells. Int Immunol 13(12):1489–1499CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Séverine Rémy
    • 1
    • 2
    • 3
  • Laurent Tesson
    • 1
    • 2
    • 3
  • Séverine Ménoret
    • 1
    • 2
    • 3
  • Claire Usal
    • 1
    • 2
    • 3
  • Andrew M. Scharenberg
    • 4
  • Ignacio Anegon
    • 1
    • 2
    • 3
    Email author
  1. 1.INSERM, U643NantesFrance
  2. 2.CHU Nantes, Institut de Transplantation et de Recherche en Transplantation, ITERTNantesFrance
  3. 3.Université de Nantes, Faculté de MédecineNantesFrance
  4. 4.Seattle Children’s Research InstituteSeattleUSA

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