Abstract
Zebrafish are a powerful model system for in vivo analysis of neural development. The degree of genetic characterization, the availability of transgenic animals, and the feasibility of in vivo imaging analysis makes zebrafish an excellent genetic model organism. In vivo electroporation has emerged as an important tool for studies examining the molecular mechanisms of neural development in zebrafish. In vivo electroporation offers precise temporal control over the induction of gain or loss of gene function because it can be used to target embryos at any developmental stage. Furthermore, in vivo electroporation allows for spatial targeting of different regions of the developing nervous system, and when combined with Gal4-based transgenic zebrafish can even target specific cell types. This chapter first outlines the basic protocol for targeting the zebrafish nervous system through electroporation, discussing the details concerning the choice of equipment, approach, and reagents. Then, a method utilizing a Gal4-based transgenic line to specifically target cells of the olfactory bulb is described in detail, and representative result are shown. Using this approach, in vivo electroporation can yield precise temporal control of genetic manipulations and can spatially target specific tissues or cell types.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Grunwald DJ, Eisen JS (2002) Headwaters of the zebrafish—emergence of a new model vertebrate. Nat Rev Genet 3:717–724
Halpern ME, Rhee J, Goll MG et al (2008) Gal4/UAS transgenic tools and their application to zebrafish. Zebrafish 5:97–110
le Trinh A, Fraser SE (2013) Enhancer and gene traps for molecular imaging and genetic analysis in zebrafish. Dev Growth Differ 55:434–445
Weber T, Köster R (2013) Genetic tools for multicolor imaging in zebrafish larvae. Methods 62:279–291
Lichtman JW, Fraser SE (2001) The neuronal naturalist: watching neurons in their native habitat. Nat Neurosci 4(Suppl):1215–1220
Muramatsu T, Mizutani Y, Ohmori Y, Okumura J (1997) Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo. Biochem Biophys Res Commun 230:376–380
Swartz M, Eberhart J, Mastick G, Krull CE (2001) Sparking new frontiers: using in vivo electroporation for genetic manipulations. Dev Biol 233:13–21
Krull CE (2004) A primer on using in ovo electroporation to analyze gene function. Dev Dyn 229:433–439
Stern CD (2005) The chick; a great model system becomes even greater. Dev Cell 8:9–17
Sauka-Spengler T, Barembaum M (2008) Gain- and loss-of-function approaches in the chick embryo. Methods Cell Biol 87:237–256
Haas K, Sin WC, Javaherian A, Li Z, Cline HT (2001) Single-cell electroporation for gene transfer in vivo. Neuron 229:583–591
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
Bestman JE, Ewald RC, Chiu S, Cline HT (2006) In vivo single-cell electroporation for transfer of DNA and macromolecules. Nat Protoc 1:1267–1272
Falk J, Drinjakovic J, Leung KM, Dwivedy A, Regan AG, Piper M, Holt CE (2007) Electroporation of cDNA/Morpholinos to targeted areas of the embryonic CNS in Xenopus. BMC Dev Biol 7:107–115
Teh C, Chong SW, Korzh V (2003) DNA delivery into anterior neural tube of zebrafish embryos by electroporation. Biotechniques 35:950–954
Teh C, Parinov S, Korzh V (2005) New ways to admire zebrafish: progress in functional genomics research methodology. Biotechniques 38:897–906
Cerda GA, Thomas JE, Allende ML, Karlstrom RO, Palma V (2006) Electroporation of DNA, RNA, and morpholinos into zebrafish embryos. Methods 39:207–211
Hendricks M, Jesuthasan S (2007) Electroporation-based methods for in vivo, whole mount and primary culture analysis of zebrafish brain development. Neural Dev 15:2–6
Kera SA, Agerwala SM, Horne JH (2010) The temporal resolution of in vivo electroporation in zebrafish: a method for time-resolved loss-of-function. Zebrafish 7:97–108
Hoegler KJ, Horne JH (2010) Targeting the zebrafish optic tectum using in vivo electroporation. Cold Spring Harb Protoc. doi:10.1101/pdb.prot5463
Hoegler KJ, Distel M, Köster RW, Horne JH (2011) Targeting olfactory bulb neurons using combined in vivo electroporation and Gal4-based enhancer trap zebrafish lines. J Vis Exp. doi:10.3791/2964
Dong Z, Wagle M, Guo S (2011) Time-lapse live imaging of clonally related neural progenitor cells in the developing zebrafish forebrain. J Vis Exp. doi:10.3791/2594
Bhatt DH, Otto SJ, Depoister B, Fetcho JR (2004) Cyclic AMP-induced repair of zebrafish spinal circuits. Science 305:254–258
Tawk M, Bianco IH, Clarke JD (2009) Focal electroporation in zebrafish embryos and larvae. Methods Mol Biol 546:145–151
Feng Y, Yan T, Zheng J et al (2010) Overexpression of Wld(S) or Nmnat2 in mauthner cells by single-cell electroporation delays axon degeneration in live zebrafish. J Neurosci Res 88:3319–3327
Kassing V, Engelmann J, Kurtz R (2013) Monitoring of single-cell responses in the optic tectum of adult zebrafish with dextran-coupled calcium dyes delivered via local electroporation. PLoS One. doi:10.1371/journal.pone.0062846
Rambabu KM, Rao SH, Rao NM (2005) Efficient expression of transgenes in adult zebrafish by electroporation. BMC Biotechnol 13:5–29
Rao NM, Rambabu KM, Rao SH (2008) Electroporation of adult zebrafish. Methods Mol Biol 423:289–298
Hyde DR, Godwin AR, Thummel R (2012) In vivo electroporation of morpholinos into the regenerating adult zebrafish tailfin. J Vis Exp. doi:10.3791/3632
McCauley DW, Bronner-Fraser M (2006) Importance of SoxE in neural crest development and the evolution of the pharynx. Nature 441:750–752
Taneyhill LA, Coles EG, Bronner-Fraser M (2007) Snail2 directly represses cadherin6B during epithelial-to-mesenchymal transitions of the neural crest. Development 134:1481–1490
Thummel R, Bailey TJ, Hyde DR (2011) In vivo electroporation of morpholinos into the adult zebrafish retina. J Vis Exp. doi:10.3791/3603
Holmes KE, Wyatt MJ, Shen YC, Thompson DA, Barald KF (2011) Direct delivery of MIF morpholinos into the zebrafish otocyst by injection and electroporation affects inner ear development. J Vis Exp. doi:10.3791/2466
Teruel MN, Meyer T (1997) Electroporation-induced formation of individual calcium entry sites in the cell body and processes of adherent cells. Biophys J 73:1785–1796
Rosen JN, Sweeney MF, Mably JD (2009) Microinjection of zebrafish embryos to analyze gene function. J Vis Exp. doi:10.3791/1115
Lucas ML, Jaroszeski MJ, Gilbert R, Heller R (2001) In vivo electroporation using an exponentially enhanced pulse: a new waveform. DNA Cell Biol 20:183–188
Borges RM, Horne JH, Melo A, Vidal JT et al (2013) A detailed description of an economical setup for electroporation of chick embryos in ovo. Braz J Med Biol Res 46:752–757
Davison JM, Akitake CM, Goll MG, Rhee JM et al (2007) Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish. Dev Biol 304:811–824
Scott EK, Mason L, Arrenberg AB, Ziv L et al (2007) Targeting neural circuitry in zebrafish using GAL4 enhancer trapping. Nat Methods 4:323–326
Distel M, Wullimann MF, Köster RW (2009) Optimized Gal4 genetics for permanent gene expression mapping in zebrafish. Proc Natl Acad Sci U S A 106:13365–13370
Nobuhiko M, Kozo M, Tatsuya T, Shin-ichi H et al (2009) From the olfactory bulb to higher brain centers: genetic visualization of secondary olfactory pathways in zebrafish. J Neurosci 29:4756–4766
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Horne, J.H. (2015). Targeting the Zebrafish Nervous System Using In Vivo Electroporation. In: Saito, T. (eds) Electroporation Methods in Neuroscience. Neuromethods, vol 102. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2459-2_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2459-2_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2458-5
Online ISBN: 978-1-4939-2459-2
eBook Packages: Springer Protocols