Electroporation in the Regenerating Tail of the Xenopus Tadpole

Xenopus laevis is a model system widely used to investigate embryogenesis, metamorphosis, and regeneration. The tail of the Xenopus tadpole is very useful in analyzing the molecular mechanisms underlying appendage regeneration (Slack et al., 2004; Mochii et al., 2007; Slack et al., 2008). It is transparent and suitable for whole-mount observation at the cellular level. The tail regenerates within 2 weeks of amputation. The conventional injection of blastomeres with mRNA, DNA, or antisense oligonucleotides is a powerful tool with which to study genetic mechanisms in early embryos, but it is not effective in late embryos or larvae. A transgenic approach has been used to analyze tail regeneration (Beck et al., 2003, 2006), but its success is largely dependent on the activity of the promoter used. There are limited numbers of promoters available that precisely regulate gene expression spatially and/or temporally. In vivo electroporation is an alternative method that can be used to manipulate gene expression in late embryos and larvae. The introduction of DNA or RNA into the cells of neurula and tailbud embryos has been reported (Eide et al., 2000; Sasagawa et al., 2002; Falk et al., 2007). Targeting larval tissues with in vivo electroporation also has been used to investigate neural networks, metamorphosis, and regeneration (Haas et al., 2001, 2002; Nakajima and Yaoita, 2003; Javaherian and Cline, 2005; Bestman et al., 2006; Boorse et al., 2006; Lin et al., 2007; Mochii et al., 2007). In this chapter, we report a procedure to introduce DNA into the tissues of the tadpole tail.


Green Fluorescent Protein Green Fluorescent Protein Fluorescence Notochord Cell Late Embryo Xenopus Tadpole 


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  1. Adams DS, Keller R, Koehl MA (1990) The mechanics of notochord elongation, straightening and stiffening in the embryo of Xenopus laevis. Development 110:115–130.Google Scholar
  2. Atkinson DL, Stevenson TJ, Park EJ, Riedy MD, Milash B, Odelberg SJ (2006) Cellular electro-poration induces dedifferentiation in intact newt limbs. Dev Biol 299:257–271.CrossRefGoogle Scholar
  3. Beck CW, Christen B, Slack JM (2003) Molecular pathways needed for regeneration of spinal cord and muscle in a vertebrate. Dev Cell 5:429–439.CrossRefGoogle Scholar
  4. Beck CW, Christen B, Barker D, Slack JM (2006) Temporal requirement for bone morphogenetic proteins in regeneration of the tail and limb of Xenopus tadpoles. Mech Dev 123:674–688.CrossRefGoogle Scholar
  5. Bestman JE, Ewald RC, Chiu SL, Cline HT (2006) In vivo single-cell electroporation for transfer of DNA and macromolecules. Nat Protoc 1:1267–1272.CrossRefGoogle Scholar
  6. Boorse GC, Kholdani CA, Seasholtz AF, Denver RJ (2006) Corticotropin-releasing factor is cytoprotective in Xenopus tadpole tail:Coordination of ligand, receptor, and binding protein in tail muscle cell survival. Endocrinology 147:1498–1507.CrossRefGoogle Scholar
  7. Echeverri K, Tanaka EM (2002) Ectoderm to mesoderm lineage switching during axolotl tail regeneration. Science 298:1993–1996.CrossRefGoogle Scholar
  8. Eide FF, Eisenberg SR, Sanders TA (2000) Electroporation-mediated gene transfer in free-swimming embryonic Xenopus laevis. FEBS Lett 486:29–32.CrossRefGoogle Scholar
  9. Falk J, Drinjakovic J, Leung KM, Dwivedy A, Regan AG, Piper M, Holt CE (2007) Electroporation of cDNA/morpholinos to targeted areas of embryonic CNS in Xenopus. BMC Dev Biol. doi:10.1186/1471-213X-7-107.Google Scholar
  10. Gargioli C, Slack JM (2004) Cell lineage tracing during Xenopus tail regeneration. Development 131:2669–2679.CrossRefGoogle Scholar
  11. González-Estévez C, Momose T, Gehring WJ, Saló E (2003) Transgenic planarian lines obtained by electroporation using transposon-derived vectors and an eye-specific GFP marker. Proc Natl Acad Sci USA 100:14046–14051.CrossRefGoogle Scholar
  12. Haas K, Sin WC, Javaherian A, Li Z, Cline HT (2001) Single-cell electroporation for gene transfer in vivo. Neuron 29:583–591.CrossRefGoogle Scholar
  13. Haas K, Jensen K, Sin WC, Foa L, Cline HT (2002) Targeted electroporation in Xenopus tadpoles in vivo — from single cells to the entire brain. Differentiation 70:148–154.CrossRefGoogle Scholar
  14. Inouye S, Ogawa H, Yasuda K, Umesono K, Tsuji FI (1997) A bacterial cloning vector using a mutated Aequorea green fluorescent protein as an indicator. Gene 189:159–162.CrossRefGoogle Scholar
  15. Javaherian A, Cline HT (2005) Coordinated motor neuron axon growth and neuromuscular syn-aptogenesis are promoted by CPG15 in vivo. Neuron 45:505–512.CrossRefGoogle Scholar
  16. Lin G, Chen Y, Slack JM (2007) Regeneration of neural crest derivatives in the Xenopus tadpole tail. BMC Dev Biol doi:10.1186/1471-213X-7-56.Google Scholar
  17. Mochii M, Taniguchi Y, Shikata I. (2007) Tail regeneration in the Xenopus tadpole. Dev Growth Differ 49:155–161.Google Scholar
  18. Nakajima K, Yaoita Y (2003) Dual mechanisms governing muscle cell death in tadpole tail during amphibian metamorphosis. Dev Dyn 227:246–255.CrossRefGoogle Scholar
  19. Nieuwkoop PD, Faber J (1994) Normal Table of Xenopus Laevis (Daudin):A Systematical and Chronological Survey of the Development from the Fertilized Egg Till the End of Metamorphosis. Garland, New York.Google Scholar
  20. Niwa H, Yamamura K, Miyazaki J (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193–199.CrossRefGoogle Scholar
  21. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning:A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  22. Sasagawa S, Takabatake T, Takabatake Y, Muramatsu T, Takeshima K (2002) Improved mRNA electroporation method for Xenopus neurula embryos. Genesis 33:81–85.CrossRefGoogle Scholar
  23. Sato Y, Kasai T, Nakagawa S, Tanabe K, Watanabe T, Kawakami K, Takahashi Y. (2007) Stable integration and conditional expression of electroporated transgenes in chicken embryos. Dev Biol 305:616–624.CrossRefGoogle Scholar
  24. Schnapp E, Tanaka EM (2005) Quantitative evaluation of morpholino-mediated protein knockdown of GFP, MSX1, and PAX7 during tail regeneration in Ambystoma mexicanum. Dev Dyn 232:162–170. Erratum in:Dev Dyn 233:1175.CrossRefGoogle Scholar
  25. Slack JM, Beck CW, Gargioli C, Christen B (2004) Cellular and molecular mechanisms of regeneration in Xenopus. Philos Trans R Soc Lond B Biol Sci 359:745–751.CrossRefGoogle Scholar
  26. Slack JM, Lin G, Chen Y (2008) Molecular and cellular basis of regeneration and tissue repair:The Xenopus tadpole:A new model for regeneration research. Cell Mol Life Sci 65:54–63.CrossRefGoogle Scholar
  27. Thummel R, Bai S, Sarras MP Jr, Song P, McDermott J, Brewer J, Perry M, Zhang X, Hyde DR, Godwin AR (2006) Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb. Dev Dyn 235:336–346.CrossRefGoogle Scholar

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© Springer 2009

Authors and Affiliations

  1. 1.Graduate School of Life ScienceUniversity of HyogoAkou-gunJapan

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