Advertisement

Recent Advances in Meganuclease-and Transposon-Mediated Transgenesis of Medaka and Zebrafish

  • Clemens Grabher
  • Joachim Wittbrodt
Part of the METHODS IN MOLECULAR BIOLOGY™ book series (MIMB, volume 461)

1. Introduction

Transgenesis, the introduction of transgenes into the genome of an organism and its appropriate expression in subsequent generations, represents a major technological advance in modern biology. Ectopic expression of transgenes (gain of function) and disruption of endogenous genes (loss of function) in trans-genic animals have proven to be highly valuable in extending our knowledge of mechanisms of development and developmental gene regulation, the action of oncogenes, and intricate cell interactions within the immune and nervous systems. By employing reporter genes under the control of specific regulatory sequences, transgenic techniques enable the functional dissection of cis-acting elements responsible for spatial and temporal gene expression patterns. In addition, tissues or cells expressing a reporter transgene can be used in cell lineage analysis and transplantation experiments. Furthermore, the transgenic technology offers exciting possibilities for generating...

Keywords

Insertional Mutagenesis Tandem Array Homing Endonuclease Genomic Insertion Medaka Embryo 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

We are grateful to our colleagues in the lab for critical and insightful input and helpful comments, to Thorsten Henrich for his pioneering experiments with SB, and to Erika Grzebisz for excellent animal husbandry. This work was supported by grants of the German Research Foundation (DFG) and the EU to Joachim Wittbrodt.

References

  1. 1.
    Gaiano N, Allende M, Amsterdam A, Kawakami K, Hopkins, N. (1996) Highly efficient germ-line transmission of proviral insertions in zebrafish. Proc Natl Acad Sci USA 93:7777–7782.CrossRefPubMedGoogle Scholar
  2. 2.
    Lin S, Gaiano N, Culp P, Burns JC, Friedmann T, Yee JK, Hopkins N (1994) Integration and germ-line transmission of a pseudotyped retroviral vector in zebrafish. Science 265:666–669.CrossRefPubMedGoogle Scholar
  3. 3.
    Linney E, Hardison NL, Lonze BE, Lyons S, DiNapoli L (1999) Transgene Expression in zebrafish: a comparison of retroviral-vector and DNA-injection approaches. Dev Biol 213:201–216.CrossRefGoogle Scholar
  4. 4.
    Hong Y, Winkler C, Schartl M (1998) Production of medakafish chimeras from a stable embryonic stem cell line. Proc Natl Acad Sci USA 95:3679–3684.CrossRefPubMedGoogle Scholar
  5. 5.
    Inoue K, Yamashita S, Hata J, Kabeno S, Asada S, Nagahisa E, Fujita T (1990) Electroporation as a new technique for producing transgenic fish. Cell Differ Dev 29:123–128.CrossRefPubMedGoogle Scholar
  6. 6.
    Lee K-Y, Huang H, Ju B, Yang Z, Lin S (2002) Cloned zebrafish by nuclear transfer from long-term-cultured cells. Nat Biotechnol 20:795–799.PubMedGoogle Scholar
  7. 7.
    Ma CG, Fan LC, Ganassin R, Bols N, Collodi P (2001) Production of zebrafish germ-line chimeras from embryo cell cultures. Proc Natl Acad Sci USA 98:2461–2466.CrossRefPubMedGoogle Scholar
  8. 8.
    Muller F, Ivics Z, Erdelyi F, Papp T, Varadi L, Horvath L, Maclean N (1992) Introducing foreign genes into fish eggs with electroporated sperm as a carrier. Molec Marine Biol Biotechnol 1:276–281.Google Scholar
  9. 9.
    Ono H, Hirose E, Miyazaki K, Yamamoto H, Matsumoto J (1997) Transgenic medaka fish bearing the mouse tyrosinase gene: expression and transmission of the transgene following electroporation of the orange-colored variant. Pigment Cell Res 10:168–175.CrossRefPubMedGoogle Scholar
  10. 10.
    Ozato K, Kondoh H, Inohara H, Iwamatsu T, Wakamatsu Y, Okada TS (1986) Production of transgenic fish: introduction and expression of chicken delta-crystallin gene in medaka embryos. Cell Differ 19:237–244.CrossRefPubMedGoogle Scholar
  11. 11.
    Sin FY, Walker SP, Symonds JE, Mukherjee UK, Khoo JG, Sin IL (2000) Electropo-ration of salmon sperm for gene transfer: efficiency, reliability, and fate of trans-gene. Molec Repro Dev 56:285–288.CrossRefGoogle Scholar
  12. 12.
    Stuart GW, McMurray JV, Westerfield M (1988) Replication, integration and stable germ-line transmission of foreignsequences injected into early zebrafish embryos. Development 103:403–412.PubMedGoogle Scholar
  13. 13.
    Sussman R (2001) Direct DNA delivery into zebrafish embryos employing tissue culture techniques. Genesis 31:1–5.CrossRefPubMedGoogle Scholar
  14. 14.
    Tawk M, Tuil D, Torrente Y, Vriz S, Paulin D (2002) High-efficiency gene transfer into adult fish: a new tool to study fin regeneration. Genesis 32:27–31.CrossRefPubMedGoogle Scholar
  15. 15.
    Wakamatsu Y, Ju BS, Pristyaznhyuk I, Niwa K, Ladygina T, Kinoshita M, Araki K, Ozato K (2001) Fertile and diploid nuclear transplants derived from embryonic cells of a small laboratory fish, medaka (Oryzias latipes). Proc Natl Acad Sci USA 98:1071–1076.CrossRefPubMedGoogle Scholar
  16. 16.
    Yamauchi M, Kinoshita M, Sasanuma M, Tsuji S, Terada MMM, Ishikawa Y (2000) Introduction of a foreign gene into medakafish using the particle gun method. J Exp Zool 287:285–293.CrossRefPubMedGoogle Scholar
  17. 17.
    Zelenin AV, Alimov AA, Barmintzev VA, Beniumov AO, Zelenina IA, Krasnov AM, Kolesnikov VA (1991) The delivery of foreign genes into fertilized fish eggs using high-velocity microprojectiles. FEBS Lett 287:118–20.CrossRefPubMedGoogle Scholar
  18. 18.
    Zhu Z Y, Sun YH (2000) Embryonic and genetic manipulation in fish. Cell Res 10:17–27.CrossRefPubMedGoogle Scholar
  19. 19.
    Chou CY, Horng LS, Tsai HJ (2001) Uniform GFP-expression in transgenic medaka (Oryzias latipes) at the F0 generation. Transgenic Res. 10:303–315.CrossRefPubMedGoogle Scholar
  20. 20.
    Grabher C, Joly JS, Wittbrodt, J. (2004b) Highly efficient zebrafish transgenesis mediated by the meganuclease I-SceI. Methods Cell Biol in press.Google Scholar
  21. 21.
    Lin S (2000) Transgenic zebrafish. Methods Molec Biol 136:375–383.Google Scholar
  22. 22.
    Thermes V, Grabher C, Ristoratore F, Bourrat F, Choulika A, Wittbrodt J, Joly J-S (2002) I-SceI meganuclease mediates highly efficient transgenesis in fish. Mech Dev 118:91–98.CrossRefPubMedGoogle Scholar
  23. 23.
    Allen ND, Cran DG, Barton SC, Hettle S, Reik W, Surani MA (1988) Trans-genes as probes for active chromosomal domains in mouse development. Nature. 333:852–855.CrossRefPubMedGoogle Scholar
  24. 24.
    Gossler A, Joyner AL, Rossant J, Skarnes WC (1989) Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science 244:463–465.CrossRefPubMedGoogle Scholar
  25. 25.
    Korn R, Schoor M, Neuhaus H, Henseling U, Soininen R, Zachgo J, Gossler A (1992) Enhancer trap integrations in mouse embryonic stem cells give rise to staining patterns in chimaeric embryos with a high frequency and detect endogenous genes. Mechan Dev 39:95–109.CrossRefGoogle Scholar
  26. 26.
    O'Kane CJ, Gehring WJ (1987) Detection in situ of genomic regulatory elements in Drosophila. Proc Natl Acad Sci USA 84:9123–9127.CrossRefPubMedGoogle Scholar
  27. 27.
    Rubin GM, Spradling AC (1982) Genetic transformation of Drosophila with trans-posable element vectors. Science 218:348–353.CrossRefPubMedGoogle Scholar
  28. 28.
    Bayer TA, Campos-Ortega JA (1992) A transgene containing lacZ is expressed in primary sensory neurons in zebrafish. Development 115:421–426.PubMedGoogle Scholar
  29. 29.
    Collas P, Alestrom P (1998) Nuclear localization signals enhance germline transmission of a transgene in zebrafish. Transgenic Res 7:303–309.CrossRefPubMedGoogle Scholar
  30. 30.
    Culp P, Nusslein-Volhard C, Hopkins N (1991) High-frequency germ-line transmission of plasmid DNA sequences injectedinto fertilized zebrafish eggs. Proc Natl Acad Sci USA 88:7953–7957.CrossRefPubMedGoogle Scholar
  31. 31.
    Lin S, Yang S, Hopkins N (1994b) lacZ expression in germline transgenic zebrafish can be detected in living embryos. Dev Biol 161:77–83.CrossRefGoogle Scholar
  32. 32.
    Stuart GW, Vielkind JR, McMurray J V, Westerfield M (1990) Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression. Development 109:577–584.PubMedGoogle Scholar
  33. 33.
    Tanaka M, Kinoshita M (2001) Recent progress in the generation of transgenic medaka (Oryzias latipes). Zoo Sci 18:615–622.CrossRefGoogle Scholar
  34. 34.
    Higashijima S, Okamoto H, Ueno N, Hotta Y, Eguchi G (1997) High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin. Dev Biol 192:289–299.CrossRefPubMedGoogle Scholar
  35. 35.
    Dorer DR, Henikoff S (1994) Expansions of transgene repeats cause heterochromatin formation and gene silencing in Drosophila. Cell 77, 993–1002.CrossRefPubMedGoogle Scholar
  36. 36.
    Garrick D, Fiering S, Martin DIK, Whitelaw E (1998) Repeat-induced gene silencing in mammals. Nat Gen 18:56–59.CrossRefGoogle Scholar
  37. 37.
    Mehtali M, LeMeur M, Lathe R (1990) The methylation-free status of a housekeeping transgene is lost at high copy number. Gene 91:179–184.CrossRefPubMedGoogle Scholar
  38. 38.
    Iyengar A, Muller F, Maclean N (1996) Regulation and expression of transgenes in fish—a review. Transgenic Res 5:147–166.CrossRefPubMedGoogle Scholar
  39. 39.
    Elgin SC (1990) Chromatin structure and gene activity. Curr Opin Cell Biol 2: 437–445.CrossRefPubMedGoogle Scholar
  40. 40.
    Noma K, Allis CD, Grewal SI (2001) Transitions in distinct histone H3 methylation patterns at the heterochromatin domain boundaries. Science 293:1150–1155.CrossRefPubMedGoogle Scholar
  41. 41.
    Davidson AE, Balciunas D, Mohn D, Shaffer J, Hermanson S, Sivasubbu S, Cliff MP, Hackett PB, Ekker SC (2003) Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon. Dev Biol 263:191–202.CrossRefPubMedGoogle Scholar
  42. 42.
    Fadool JM, Hartl DL, Dowling JE (1998) Transposition of the mariner element from Drosophila mauritiana in zebrafish. Proc Natl Acad Sci USA 95:5182–5186.CrossRefPubMedGoogle Scholar
  43. 43.
    Grabher C, Henrich T, Sasado T, Arenz A, Wittbrodt J, Furutani-Seiki M (2003) Transposon-mediated enhancer trapping in medaka. Gene 322:57–66.CrossRefPubMedGoogle Scholar
  44. 44.
    Grabher C, Henrich, T, Sasado T, Arenz A, Wittbrodt J, Furutani-Seiki M (2004) Erratum: transposon-mediated enhancer trapping in medaka. Gene 327:239.CrossRefGoogle Scholar
  45. 45.
    Kawakami K, Shima A, Kawakami N (2000) Identification of a functional trans-posase of the Tol 2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage. Proc Natl Acad Sci USA 97:11403–11408.CrossRefPubMedGoogle Scholar
  46. 46.
    Raz E, van Luenen HG, Schaerringer B, Plasterk RHA, Driever W (1998) Transposition of the nematode Caenorhabditis elegans Tc3 element in the zebrafish Danio rerio. Curr Biol 8:82–88.CrossRefPubMedGoogle Scholar
  47. 47.
    Beylot B, Spassky A (2001) Chemical probing shows that the intron-encoded endo-nuclease I-SceI distorts DNA through binding in monomeric form to its homing site. J BiolChem 276:25243–2553.Google Scholar
  48. 48.
    Jacquier A, Dujon B (1985) An intron-encoded protein is active in a gene conversion process that spreads an intron into a mitochondrial gene. Cell 41:383–394.CrossRefPubMedGoogle Scholar
  49. 49.
    Belfort M, Roberts RJ (1997) Homing endonucleases: keeping the house in order. Nucleic Acids Res 25:3379–3388.CrossRefPubMedGoogle Scholar
  50. 50.
    Mueller JE, Bryk M, Loizos N, Belfort M (1993) In: Linn SM et al. (eds.) Nucleases. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 111–143.Google Scholar
  51. 51.
    Monteilhet C, Perrin A, Thierry A, Colleaux L, Dujon B (1990) Purification and characterization of the in vitro activity of I-Sce I, a novel and highly specific endo-nuclease encoded by a group I intron. Nucleic Acids Res18:1407–1413.CrossRefPubMedGoogle Scholar
  52. 52.
    Perrin A, Buckle M, Dujon B (1993) Asymmetrical recognition and activity of the I-SceI endonuclease on its site and on intron-exon junctions. EMBO J 12:2939–2947.PubMedGoogle Scholar
  53. 53.
    Moure CM, Gimble FS, Quiocho, F.A. (2003) The crystal structure of the gene targeting homing endonuclease I-SceI reveals the origins of its target site specificity. J Mol Biol 334:685–695.CrossRefPubMedGoogle Scholar
  54. 54.
    Choulika A, Perrin A, Dujon B, Nicolas JF (1995) Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol Cell Biol 15:1968–1973.PubMedGoogle Scholar
  55. 55.
    Bellen HJ, O'Kane CJ, Wilson C, Grossniklaus U, Pearson RK, Gehring WJ (1989) P-element-mediated enhancer detection: a versatile method to study development in Drosophila. Genes Dev 3:1288–1300.CrossRefPubMedGoogle Scholar
  56. 56.
    Spradling AC, Stern DM, Kiss I, Roote J, Laverty T, Rubin GM (1995) Gene disruptions using P transposable elements: an integral component of the Drosophila genome project. Proc Natl Acad Sci USA 92:10824–1030.CrossRefPubMedGoogle Scholar
  57. 57.
    Greenwald I (1985) lin-12, a nematode homeotic gene, is homologous to a set of mammalian proteins that includes epidermal growth factor. Cell 43:583–590.CrossRefPubMedGoogle Scholar
  58. 58.
    Moerman DG, Benian GM, Waterston RH (1986) Molecular cloning of the muscle gene unc-22 in Caenorhabditis elegans by Tc1 transposon tagging. Proc Natl Acad Sci USA 83:2579–2583.CrossRefPubMedGoogle Scholar
  59. 59.
    Osborne BI, Wirtz U, Baker B (1995) A system for insertional mutagenesis and chromosomal rearrangement using the Ds transposon and Cre-lox. Plant J 7:687–701.CrossRefPubMedGoogle Scholar
  60. 60.
    Bellen HJ, Wilson C, Gibson G, Grossniklaus U, Pearson RK, O'Kane C, Gehring WJ (1990) P-element-mediated enhancer detection allows rapid identification of developmentally regulated genes and cell specific markers in Drosophila. J Physiol (Paris) 84:33–41.Google Scholar
  61. 61.
    Cooley L, Berg C, Spradling, A. (1988) Controlling P element insertional mutagen-esis. Trends Genet 4:254–258.CrossRefPubMedGoogle Scholar
  62. 62.
    Sentry JW, Kaiser K (1992) P element transposition and targeted manipulation of the Drosophila genome. Trends Genet 8:329–331.PubMedGoogle Scholar
  63. 63.
    Gibbs PD, Gray A, Thorgaard G (1994) Inheritance of P element and reporter gene sequences in zebrafish. Mol Marine Biol Biotechnol 3:317–326.PubMedGoogle Scholar
  64. 64.
    Handler AM, Gomez SP, O'Brochta DA (1993) A functional analysis of the P-ele-ment gene-transfer vector in insects. Arch Insect Biochem Physiol 22:373–384.CrossRefPubMedGoogle Scholar
  65. 65.
    Rio DC, Barnes G, Laski FA, Rine J, Rubin GM (1988) Evidence for Drosophila P element transposase activity in mammalian cells and yeast. J Mol Biol 200:411–415.CrossRefPubMedGoogle Scholar
  66. 66.
    Calvi BR, Hong TJ, Findley SD, Gelbart WM (1991) Evidence for a common evolutionary origin of inverted repeat transposons in Drosophila and plants: hobo, Activator, and Tam 3. Cell 66:465–471.CrossRefPubMedGoogle Scholar
  67. 67.
    Koga A, Inagaki H, Bessho Y, Hori H (1995) Insertion of a novel transposable element in the tyrosinase gene is responsible for an albino mutation in the medaka fish, Oryzias latipes. Molec Genl Genet 249:400–405.Google Scholar
  68. 68.
    Koga A, Suzuki M, Inagaki H, Bessho Y, Hori H (1996) Transposable element in fish. Nature 383:30.CrossRefPubMedGoogle Scholar
  69. 69.
    Kawakami K, Noda T (2004) Transposition of the Tol 2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells. Genetics 166:895–899.CrossRefPubMedGoogle Scholar
  70. 70.
    Plasterk RH (1996) The Tc1/mariner transposon family. Curr Topics Microbiol Immunol 204:125–143.Google Scholar
  71. 71.
    Lampe DJ, Churchill ME, Robertson HM. (1996) A purified mariner transposase is sufficient to mediate transposition in vitro. EMBO J 15:5470–5479.PubMedGoogle Scholar
  72. 72.
    Vos JC, De Baere I, Plasterk RH (1996) Transposase is the only nematode protein required for in vitro transposition of Tc1. Genes Dev 10:755–761.CrossRefPubMedGoogle Scholar
  73. 73.
    Loukeris TG, Arca B, Livadaras I, Dialektaki G, Savakis C (1995) Introduction of the transposable element Minos into the germ line of Drosophila melanogaster. Proc Natl Acad Sci USA 92:9485–9489.CrossRefPubMedGoogle Scholar
  74. 74.
    Gueiros-Filho FJ, Beverley SM (1997) Trans-kingdom transposition of the Dro-sophila element mariner within the protozoan Leishmania. Science 276:1716–1719.CrossRefPubMedGoogle Scholar
  75. 75.
    Goodier JL, Davidson WS (1994) Tc1 transposon-like sequences are widely distributed in salmonids. J Mol Biol 241: 26–34.CrossRefPubMedGoogle Scholar
  76. 76.
    Ivics Z, Izsvak Z, Minter A, Hackett PB (1996) Identification of functional domains and evolution of Tc1-like transposable elements. Proc Natl Acad Sci USA 93:5008–5013.CrossRefPubMedGoogle Scholar
  77. 77.
    Lam WL, Lee TS, Gilbert W (1996a) Active transposition in zebrafish. Proc Natl Acad Sci USA 93:10870–10875.CrossRefGoogle Scholar
  78. 78.
    Lam WL, Seo P, Robison K, Virk S, Gilbert W (1996b) Discovery of amphibian Tc1-like transposon families. J Mol Biol 257:359–366.CrossRefGoogle Scholar
  79. 79.
    Radice AD, Bugaj B, Fitch DH, Emmons SW (1994) Widespread occurrence of the Tc1 transposon family: Tc1-like transposons from teleost fish. Mol Gen Genet 244:606–612.CrossRefPubMedGoogle Scholar
  80. 80.
    Ivics Z, Hackett PB, Plasterk RH, Izsvak Z (1997) Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91:501 - 5–10.Google Scholar
  81. 81.
    Dupuy AJ, Clark K, Carlson CM, Fritz S, Davidson AE, Markley KM, Finley K, Fletcher CF, Ekker SC, Hackett PB, Horn S, Largaespada DA (2002) Mammalian germ-line transgenesis by transposition. Proc Natl Acad Sci USA 99:4495–4499.CrossRefPubMedGoogle Scholar
  82. 82.
    Dupuy AJ, Fritz S, Largaespada DA (2001) Transposition and gene disruption in the male germline of the mouse. Genesis 30:82–88.CrossRefPubMedGoogle Scholar
  83. 83.
    Fischer SEJ, Wienholds E, Plasterk RHA (2001) Regulated transposition of a fish transposon in the mouse germ line. Proc Natl Acad Sci USA 98:6759–6764.CrossRefPubMedGoogle Scholar
  84. 84.
    Horie K, Kuroiwa A, Ikawa M, Okabe M, Kondoh G, Matsuda Y, Takeda J (2001) Efficient chromosomal transposition of a Tc1/mariner-like transposon Sleeping Beauty in mice. Proc Natl Acad Sci USA 98:9191–9196.CrossRefPubMedGoogle Scholar
  85. 85.
    Vigdal TJ, Kaufman CD., Izsvak Z, Voytas DF, Ivics Z (2002) Common physical properties of DNA affecting target site selection of Sleeping Beauty and other Tc1/ mariner transposable elements. J Mol Biol 323:441–452.CrossRefPubMedGoogle Scholar
  86. 86.
    Cui Z, Geurts AM, Liu G, Kaufman CD, Hackett PB (2002) Structure-function analysis of the inverted terminal repeats of the Sleeping Beauty transposon. J Mol Biol 318:1221–1235.CrossRefPubMedGoogle Scholar
  87. 87.
    Fu Y, Wang Y, Evans SM (1998) Viral sequences enable efficient and tissue-specific expression of transgenes in Xenopus. Nat Biotechnol 16:253–257.CrossRefPubMedGoogle Scholar
  88. 88.
    Hsiao CD, Hsieh FJ, Tsai HJ (2001) Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno-associated virus in zebrafish. Dev Dyn 220:323–336.CrossRefPubMedGoogle Scholar
  89. 89.
    Gong WJ, Golic KG (2003) Ends-out, or replacement, gene targeting in Drosophila. Proc Natl Acad Sci USA (in press).Google Scholar
  90. 90.
    Zayed H, Izsvak Z, Walisko O, Ivics Z (2004) Development of hyperactive Sleeping Beauty transposon vectors by mutational analysis. Mol Ther 9:292–304.CrossRefPubMedGoogle Scholar
  91. 91.
    Yusa K, Takeda J, Horie K (2004) Enhancement of Sleeping Beauty transposition by CpG methylation: possible role of heterochromatin formation. Mol Cell Biol 24:4004–4018.CrossRefPubMedGoogle Scholar
  92. 92.
    Miskey C, Izsvak Z, Plasterk RH, Ivics Z (2003) The Frog Prince: a reconstructed transposon from Rana pipiens with high transpositional activity in vertebrate cells. Nucleic Acids Res 31:6873–6881.CrossRefPubMedGoogle Scholar
  93. 93.
    Westerfield M (1995) The zebrafish book. University of Oregon Press, Eugene.Google Scholar
  94. 94.
    Yamamoto T (1975) Medaka (killifish), biology and strains. Keigaku, Tokyo.Google Scholar
  95. 95.
    Meng A, Jessen JR, Lin S (1999) Transgenesis. Methods Cell Biol 60:133–148.CrossRefPubMedGoogle Scholar
  96. 96.
    Hyatt TM, Ekker SC (1999) Vectors and techniques for ectopic gene expression in zebrafish. Methods Cell Biol 59:117–126.CrossRefPubMedGoogle Scholar
  97. 97.
    Wormington M (1991) Preparation of synthetic mRNAs and analyses of translational efficiency in microinjected Xenopus oocytes. Methods Cell Biol 36:167–183.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Clemens Grabher
    • 1
  • Joachim Wittbrodt
    • 1
  1. 1.Developmental Biology ProgramEuropean Molecular Biology Laboratory (EMBL)HeidelbergGermany

Personalised recommendations