Electroporation in Ascidians: History, Theory and Protocols

  • Robert W. ZellerEmail author
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1029)


Embryonic development depends on the orchestration of hundreds of regulatory and structural genes to initiate expression at the proper time, in the correct spatial domain(s), and in the amounts required for cells and tissues to become specified, determined, and ultimately to differentiate into a multicellular embryo. One of the key approaches to studying embryonic development is the generation of transgenic animals in which recombinant DNA molecules are transiently or stably introduced into embryos to alter gene expression, to manipulate gene function or to serve as reporters for specific cell types or subcellular compartments. In some model systems, such as the mouse, well-defined approaches for generating transgenic animals have been developed. In other systems, particularly non-model systems, a key challenge is to find a way of introducing molecules or other reagents into cells that produces large numbers of embryos with a minimal effect on normal development. A variety of methods have been developed, including the use of viral vectors, microinjection, and electroporation. Here, I describe how electroporation was adapted to generate transgenic embryos in the ascidian, a nontraditional invertebrate chordate model that is particularly well-suited for studying gene regulatory activity during development. I present a review of the electroporation process, describe how electroporation was first implemented in the ascidian, and provide a series of protocols describing the electroporation process, as implemented in our laboratory.


Electroporation Ciona Ascidiella Ascidia Molgula Phallusia Boltenia Ascidian Transgenic embryo Exponentially decaying pulse Square wave pulse 


  1. Arezzo F (1989) Sea urchin sperm as a vector of foreign genetic information. Cell Biol Int Rep 13(4):391–404CrossRefGoogle Scholar
  2. Borei HG, Bjorklund U (1953) Effect of versene on sea urchin eggs. Exp Cell Res 5(1):216–219CrossRefPubMedGoogle Scholar
  3. Brinster RL, Sandgren EP et al (1989) No simple solution for making transgenic mice. Cell 59(2):239–241CrossRefPubMedGoogle Scholar
  4. Brown CD, Johnson DS et al (2007) Functional architecture and evolution of transcriptional elements that drive gene coexpression. Science 317(5844):1557–1560CrossRefPubMedGoogle Scholar
  5. Bullmann T, Arendt T et al (2015) A transportable, inexpensive electroporator for in utero electroporation. Dev Growth Differ 57(5):369–377Google Scholar
  6. Cerda GA, Thomas JE et al (2006) Electroporation of DNA, RNA, and morpholinos into zebrafish embryos. Methods 39(3):207–211CrossRefPubMedGoogle Scholar
  7. Chen JS, Pedro MS et al (2011) miR-124 function during Ciona intestinalis neuronal development includes extensive interaction with the Notch signaling pathway. Development 138(22):4943–4953CrossRefPubMedGoogle Scholar
  8. Christiaen L, Wagner E, et al (2009a) Electroporation of transgenic DNAs in the sea squirt Ciona. Cold Spring Harb Protoc 2009(12):pdb prot5345Google Scholar
  9. Christiaen L, Wagner E et al (2009b) Microinjection of morpholino oligos and RNAs in sea squirt (Ciona) embryos. Cold Spring Harb Protoc 2009(12):pdb prot5347Google Scholar
  10. Corbo JC, Levine M et al (1997) Characterization of a notochord-specific enhancer from the Brachyury promoter region of the ascidian, Ciona Intestinalis. Development 124(3):589–602PubMedGoogle Scholar
  11. Crowther RJ, Whittaker JR (1983) Developmental autonomy of muscle fine structure in muscle lineage cells of ascidian embryos. Dev Biol 96(1):1–10CrossRefPubMedGoogle Scholar
  12. Di Gregorio AD, Levine M (2002) Analyzing gene regulation in ascidian embryos: new tools for new perspectives. Differentiation 70(4–5):132–139CrossRefPubMedGoogle Scholar
  13. Erives A, Corbo JC et al (1998) Lineage-specific regulation of the Ciona snail gene in the embryonic mesoderm and neuroectoderm. Dev Biol 194(2):213–225CrossRefPubMedGoogle Scholar
  14. Escoffre JM, Portet T et al (2009) What is (still not) known of the mechanism by which electroporation mediates gene transfer and expression in cells and tissues. Mol Biotechnol 41(3):286–295CrossRefPubMedGoogle Scholar
  15. Falk J, Drinjakovic J et al (2007) Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus. BMC Dev Biol 7:107CrossRefPubMedPubMedCentralGoogle Scholar
  16. Flytzanis CN, McMahon AP et al (1985) Persistence and integration of cloned DNA in postembryonic sea urchins. Dev Biol 108(2):431–442CrossRefPubMedGoogle Scholar
  17. Franks RR, Hough-Evans BR et al (1988) Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA. Development 102(2):287–299PubMedGoogle Scholar
  18. Golzio M, Teissie J et al (2002) Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci U S A 99(3):1292–1297CrossRefPubMedPubMedCentralGoogle Scholar
  19. Hikosaka A, Kusakabe T et al (1992) Introduction and expression of recombinant genes in ascidian embryos. Develop Growth Differ 34(6):627–634CrossRefGoogle Scholar
  20. Hough-Evans BR, Britten RJ et al (1988) Mosaic incorporation and regulated expression of an exogenous gene in the sea urchin embryo. Dev Biol 129(1):198–208CrossRefPubMedGoogle Scholar
  21. Inoue K, Yamashita S et al (1990) Electroporation as a new technique for producing transgenic fish. Cell Differ Dev 29(2):123–128CrossRefPubMedGoogle Scholar
  22. Johnson DS, Davidson B et al (2004) Noncoding regulatory sequences of Ciona exhibit strong correspondence between evolutionary constraint and functional importance. Genome Res 14(12):2448–2456CrossRefPubMedPubMedCentralGoogle Scholar
  23. Kari W, Zeng F et al (2016) Embryo microinjection and electroporation in the Chordate Ciona intestinalis. J Vis Exp 2016(116)Google Scholar
  24. Lavitrano M, Camaioni A et al (1989) Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice. Cell 57(5):717–723CrossRefPubMedGoogle Scholar
  25. Livant DL, Hough-Evans BR et al (1991) Differential stability of expression of similarly specified endogenous and exogenous genes in the sea urchin embryo. Development 113(2):385–398PubMedGoogle Scholar
  26. Matsumoto J, Dewar K et al (2010) High-throughput sequence analysis of Ciona intestinalis SL trans-spliced mRNAs: alternative expression modes and gene function correlates. Genome Res 20(5):636–645CrossRefPubMedPubMedCentralGoogle Scholar
  27. Matsuoka T, Awazu S et al (2005) Germline transgenesis of the ascidian Ciona intestinalis by electroporation. Genesis 41(2):67–72CrossRefPubMedGoogle Scholar
  28. McDougall A, Levasseur M (1998) Sperm-triggered calcium oscillations during meiosis in ascidian oocytes first pause, restart, then stop: correlations with cell cycle kinase activity. Development 125(22):4451–4459PubMedGoogle Scholar
  29. McDougall A, Lee KW et al (2014) Microinjection and 4D fluorescence imaging in the eggs and embryos of the ascidian Phallusia mammillata. Methods Mol Biol 1128:175–185CrossRefPubMedGoogle Scholar
  30. Mita-Miyazawa I, Ikegami S et al (1985) Histospecific acetylcholinesterase development in the presumptive muscle cells isolated from 16-cell-stage ascidian embryos with respect to the number of DNA replications. J Embryol Exp Morphol 87:1–12PubMedGoogle Scholar
  31. Muller F, Ivics Z et al (1992) Introducing foreign genes into fish eggs with electroporated sperm as a carrier. Mol Mar Biol Biotechnol 1(4–5):276–281PubMedGoogle Scholar
  32. Neumann E, Schaefer-Ridder M et al (1982) Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1(7):841–845PubMedPubMedCentralGoogle Scholar
  33. Nishida H (1987) Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. III. Up to the tissue restricted stage. Dev Biol 121(2):526–541CrossRefPubMedGoogle Scholar
  34. Nishida H (1992) Regionality of egg cytoplasm that promotes muscle differentiation in embryo of the ascidian, Halocynthia roretzi. Development 116(3):521–529Google Scholar
  35. Nishida H, Satoh N (1983) Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. I. Up to the eight-cell stage. Dev Biol 99(2):382–394CrossRefPubMedGoogle Scholar
  36. Nishida H, Satoh N (1985) Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. II. The 16- and 32-cell stages. Dev Biol 110(2):440–454CrossRefPubMedGoogle Scholar
  37. Okkema PG, Harrison SW et al (1993) Sequence requirements for myosin gene expression and regulation in Caenorhabditis elegans. Genetics 135(2):385–404PubMedPubMedCentralGoogle Scholar
  38. Phez E, Faurie C et al (2005) New insights in the visualization of membrane permeabilization and DNA/membrane interaction of cells submitted to electric pulses. Biochim Biophys Acta 1724(3):248–254CrossRefPubMedGoogle Scholar
  39. Powers DA, Hereford L et al (1992) Electroporation: a method for transferring genes into the gametes of zebrafish (Brachydanio rerio), channel catfish (Ictalurus punctatus), and common carp (Cyprinus carpio). Mol Mar Biol Biotechnol 1(4–5):301–308PubMedGoogle Scholar
  40. Powers DA, Kirby VL et al (1995) Electroporation as an effective means of introducing DNA into abalone (Haliotis rufescens) embryos. Mol Mar Biol Biotechnol 4(4):369–375PubMedGoogle Scholar
  41. Prodon F, Chenevert J et al (2010) Dual mechanism controls asymmetric spindle position in ascidian germ cell precursors. Development 137(12):2011–2021CrossRefPubMedGoogle Scholar
  42. Roure A, Lemaire P et al (2014) An otx/nodal regulatory signature for posterior neural development in ascidians. PLoS Genet 10(8):e1004548CrossRefPubMedPubMedCentralGoogle Scholar
  43. Sardet C, McDougall A et al (2011) Embryological methods in ascidians: the Villefranche-sur-Mer protocols. Methods Mol Biol 770:365–400CrossRefPubMedGoogle Scholar
  44. Sato Y, Morisawa M (1999) Loss of test cells leads to the formation of new tunic surface cells and abnormal metamorphosis in larvae of Ciona intestinalis (Chordata, ascidiacea). Dev Genes Evol 209(10):592–600CrossRefPubMedGoogle Scholar
  45. Satou Y, Imai KS et al (2001) Action of morpholinos in Ciona embryos. Genesis 30(3):103–106CrossRefPubMedGoogle Scholar
  46. Smith K, Spadafora C (2005) Sperm-mediated gene transfer: applications and implications. BioEssays 27(5):551–562CrossRefPubMedGoogle Scholar
  47. Stinchcomb DT, Shaw JE et al (1985) Extrachromosomal DNA transformation of Caenorhabditis elegans. Mol Cell Biol 5(12):3484–3496CrossRefPubMedPubMedCentralGoogle Scholar
  48. Stolfi A, Christiaen L (2012) Genetic and genomic toolbox of the chordate Ciona intestinalis. Genetics 192(1):55–66CrossRefPubMedPubMedCentralGoogle Scholar
  49. Stolfi A, Lowe EK et al (2014) Divergent mechanisms regulate conserved cardiopharyngeal development and gene expression in distantly related ascidians. elife 3:e03728CrossRefPubMedPubMedCentralGoogle Scholar
  50. Symonds JE, Walker SP et al (1994) Electroporation of salmon sperm with plasmid DNA: evidence of enhanced sperm/DNA association. Aquaculture 119(4):313–327CrossRefGoogle Scholar
  51. Vierra DA, Irvine SQ (2012) Optimized conditions for transgenesis of the ascidian Ciona using square wave electroporation. Dev Genes Evol 222(1):55–61CrossRefPubMedGoogle Scholar
  52. Zeller RW (2004) Generation and use of transgenic ascidians. In: Ettensohn C, Wray G, Wessels G (eds) Experimental analysis of the development of sea urchins and other non-vertebrate deuterostomes. AcademicGoogle Scholar
  53. Zeller RW, Virata MJ et al (2006) Predictable mosaic transgene expression in ascidian embryos produced with a simple electroporation device. Dev Dyn 235(7):1921–1932CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Center for Applied and Experimental Genomics, Department of BiologySan Diego State UniversitySan DiegoUSA

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