Temporal and Tissue-Specific Control of Gene Expression in the Peri-Implantation Mouse Embryo Through Electroporation of dsRNA

  • Miguel L. Soares
  • Maria-Elena Torres-Padilla

The delivery of nucleic acids into embryos — either DNA molecules for transient expression or double-stranded RNA for gene silencing by RNA interference (RNAi) — remains a challenging aspect of functional studies on live organisms. Electroporation has long been a standard method for the active transfer of the nega tively charged nucleic acids into mammalian cells (Andreason and Evans, 1988). This technique employs electric pulses to create transient pores in the cytoplasmic membrane through which the nucleic acids are actively delivered. It was not until the conditions for culture of whole embryos became consistent, however, that it has been applied successfully for transfection of mouse concepti.

Nucleic acids delivery by electroporation has been achieved at various stages of mouse embryonic development. Conditions for successful electroporation in pre implantation stages have been established and optimized allowing whole-embryo tar geting of zygotes, morulae and blastocysts (Grabarek et al., 2002; Soares et al., 2005). Post-implantation embryos have undergone the procedure from as early as embryonic day (E) 5.25 up to E13.0 (Calegari et al., 2002; Mellitzer et al., 2002; Holm et al., 2007; Soares et al., 2008). This temporal specificity is particularly useful when study ing signaling pathways at work within relatively small time windows in development. In early post-implantation development, for instance, just before gastrulation, signal ing events shaping the establishment of the body axes of the mouse conceptus have been studied by temporal-specific electroporation of dsRNA (Soares et al., 2008).


Inner Cell Mass Preimplantation Embryo Visceral Endoderm Parietal Endoderm Extraembryonic Ectoderm 
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.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Andreason GL, Evans GA (1988) Introduction and expression of DNA molecules in eukaryotic cells by electroporation. Biotechniques 6:650–660.Google Scholar
  2. Calegari F, Haubensak W, Yang D, Huttner WB, Buchholz F (2002) Tissue-specific RNA inter ference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA. Proc Natl Acad Sci U S A 99:14236–14240.CrossRefGoogle Scholar
  3. Chen G, Sima J, Jin M, Wang KY, Xue XJ, Zheng W, Ding YQ, Yuan XB (2008) Semaphorin-3A guides radial migration of cortical neurons during development. Nat Neurosci 11:36–44.CrossRefGoogle Scholar
  4. Coucouvanis E, Martin GR (1999) BMP signaling plays a role in visceral endoderm differentia tion and cavitation in the early mouse embryo. Development 126:535–546.Google Scholar
  5. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498.CrossRefGoogle Scholar
  6. Erbach GT, Lawitts JA, Papaioannou VE, Biggers JD (1994) Differential growth of the mouse preimplantation embryo in chemically defined media. Biol Reprod 50:1027–1033.CrossRefGoogle Scholar
  7. Fujinaga M (2000) In vitro culture of rodent embryos during the early postimplantation period. Methods Mol Biol 135:53–76.Google Scholar
  8. Garcia-Frigola C, Carreres MI, Vegar C, Herrera E (2007) Gene delivery into mouse retinal gan glion cells by in utero electroporation. BMC Dev Biol 7:103.CrossRefGoogle Scholar
  9. Grabarek JB, Plusa B, Glover DM, Zernicka-Goetz M (2002) Efficient delivery of dsRNA into zona-enclosed mouse oocytes and preimplantation embryos by electroporation. Genesis 32:269–276.CrossRefGoogle Scholar
  10. Hadjantonakis AK, Papaioannou VE (2004) Dynamic in vivo imaging and cell tracking using a histone fluorescent protein fusion in mice. BMC Biotechnol 4:33.CrossRefGoogle Scholar
  11. Hamblet NS, Lijam N, Ruiz-Lozano P, Wang J, Yang Y, Luo Z, Mei L, Chien KR, Sussman DJ, Wynshaw-Boris A (2002) Dishevelled 2 is essential for cardiac outflow tract development, somite segmentation and neural tube closure. Development 129:5827–5838.CrossRefGoogle Scholar
  12. Hogan BL (1996) Bone morphogenetic proteins:multifunctional regulators of vertebrate develop ment. Genes Dev 10:1580–1594.CrossRefGoogle Scholar
  13. Holm PC, Mader MT, Haubst N, Wizenmann A, Sigvardsson M, Götz M (2007) Loss- and gain-of-function analyses reveal targets of Pax6 in the developing mouse telencephalon. Mol Cell Neurosci 34:99–119.CrossRefGoogle Scholar
  14. Jones EA, Crotty D, Kulesa PM, Waters CW, Baron MH, Fraser SE, Dickinson ME (2002) Dynamic in vivo imaging of postimplantation mammalian embryos using whole embryo cul ture. Genesis 34:228–235.CrossRefGoogle Scholar
  15. Kelly OG, Pinson KI, Skarnes WC (2004) The Wnt co-receptors Lrp5 and Lrp6 are essential for gastrulation in mice. Development 131:2803–2815.CrossRefGoogle Scholar
  16. Lawitts JA, Biggers JD (1991) Optimization of mouse embryo culture media using simplex meth ods. J Reprod Fertil 91:543–556.Google Scholar
  17. Lawitts JA, Biggers JD (1993) Culture of preimplantation embryos. Methods Enzymol 225:153–164.CrossRefGoogle Scholar
  18. Lee SB, Esteban M (1994) The interferon-induced double-stranded RNA-activated protein kinase induces apoptosis. Virology 199:491–496.CrossRefGoogle Scholar
  19. Lijam N, Paylor R, McDonald MP, Crawley JN, Deng CX, Herrup K, Stevens KE, Maccaferri G, McBain CJ, Sussman DJ, Wynshaw-Boris A (1997) Social interaction and sensorimotor gating abnormalities in mice lacking Dvl1. Cell 90:895–905.CrossRefGoogle Scholar
  20. Liu P, Wakamiya M, Shea MJ, Albrecht U, Behringer RR, Bradley A (1999) Requirement for Wnt3 in vertebrate axis formation. Nat Genet 22:361–365.CrossRefGoogle Scholar
  21. Mellitzer G, Hallonet M, Chen L, Ang SL (2002) Spatial and temporal ‘knock down’ of gene expression by electroporation of double-stranded RNA and morpholinos into early postim-plantation mouse embryos. Mech Dev 118:57–63.CrossRefGoogle Scholar
  22. Mesnard D, Filipe M, Belo JA, Zernicka-Goetz M (2004) The anterior-posterior axis emerges respecting the morphology of the mouse embryo that changes and aligns with the uterus before gastrulation. Curr Biol 14:184–196.Google Scholar
  23. Moon RT, Brown JD, Torres M (1997) WNTs modulate cell fate and behavior during vertebrate development. Trends Genet 13:157–162.CrossRefGoogle Scholar
  24. Muramatsu T, Mizutani Y, Ohmori Y, Okumura J (1997) Comparison of three nonviral transfec-tion methods for foreign gene expression in early chicken embryos in ovo. Biochem Biophys Res Commun 230:376–380.CrossRefGoogle Scholar
  25. Nicolson GL, Yanagimachi R, Yanagimachi H (1975) Ultrastructural localization of lectin-binding sites on the zonae pellucidae and plasma membranes of mammalian eggs. J Cell Biol 66:263–274.CrossRefGoogle Scholar
  26. Perea-Gomez A, Meilhac SM, Piotrowska-Nitsche K, Gray D, Collignon J, Zernicka-Goetz M (2007) Regionalization of the mouse visceral endoderm as the blastocyst transforms into the egg cylinder. BMC Dev Biol 7:96.CrossRefGoogle Scholar
  27. Quinn P, Barros C, Whittingham DG (1982) Preservation of hamster oocytes to assay the fertiliz ing capacity of human spermatozoa. J Reprod Fertil 66:161–168.Google Scholar
  28. Rhinn M, Dierich A, Shawlot W, Behringer RR, Le Meur M, Ang SL (1998) Sequential roles for Otx2 in visceral endoderm and neuroectoderm for forebrain and midbrain induction and specification. Development 125:845–856.Google Scholar
  29. Richardson L, Torres-Padilla ME, Zernicka-Goetz M (2006) Regionalised signalling within the extraembryonic ectoderm regulates anterior visceral endoderm positioning in the mouse embryo. Mech Dev 123:288–296.CrossRefGoogle Scholar
  30. 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
  31. Soares ML, Haraguchi S, Torres-Padilla ME, Kalmar T, Carpenter L, Bell G, Morrison A, Ring CJ, Clarke NJ, Glover DM, Zernicka-Goetz M (2005) Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi. BMC Dev Biol 5:28.CrossRefGoogle Scholar
  32. Soares ML, Torres-Padilla ME, Zernicka-Goetz M (2008) BMP4 signaling regulates development of the anterior visceral endoderm in the mouse embryo. Dev Growth Differ 50:615–621.CrossRefGoogle Scholar
  33. Srinivas S, Rodriguez T, Clements M, Smith JC, Beddington RS (2004) Active cell migration drives the unilateral movements of the anterior visceral endoderm. Development 131(5):1157–1164.CrossRefGoogle Scholar
  34. Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD (1998) How cells respond to interferons. Annu Rev Biochem 67:227–264.CrossRefGoogle Scholar
  35. Summers MC, Bhatnagar PR, Lawitts JA, Biggers JD (1995) Fertilization in vitro of mouse ova from inbred and outbred strains:complete preimplantation embryo development in glucose-supplemented KSOM. Biol Reprod 53:431–437.CrossRefGoogle Scholar
  36. Svoboda P, Stein P, Hayashi H, Schultz RM (2000) Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development 127:4147–4156.Google Scholar
  37. Takahashi M, Nomnura T, Osumi N (2008) Transferring genes into cultured mammalian embryos by electroporation. Dev Growth Differ 50:485–497.CrossRefGoogle Scholar
  38. Tam PP (1998) Postimplantation mouse development:whole embryo culture and micro-manipulation. Int J Dev Biol 42:895–902.Google Scholar
  39. Tam PP, Snow MH (1980) The in vitro culture of primitive-streak-stage mouse embryos. J Embryol Exp Morphol 59:131–143.Google Scholar
  40. Torres-Padilla ME, Richardson L, Kolasinska P, Meilhac SM, Luetke-Eversloh M V, Zernicka-Goetz M (2007) The anterior visceral endoderm of the mouse embryo is established from both preimplantation precursor cells and by de novo gene expression after implantation. Dev Biol 309:97–112.CrossRefGoogle Scholar
  41. Wang J, Hamblet NS, Lijam N, Sussman DJ, Wynshaw-Boris A (2004a) The murine Dishevelled 2 gene is essential for neural tube closure [abstract]. Mouse Genetics Meeting. Cold Spring Harbor. pp. 200.Google Scholar
  42. Wang QT, Piotrowska K, Ciemerych MA, Milenkovic L, Scott MP, Davis RW, Zernicka-Goetz M (2004b) A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo. Dev Cell 6:133–144.CrossRefGoogle Scholar
  43. Wianny F, Zernicka-Goetz M (2000) Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Biol 2:70–75.CrossRefGoogle Scholar
  44. Winnier G, Blessing M, Labosky PA, Hogan BL (1995) Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev 9:2105–2116.CrossRefGoogle Scholar
  45. Yamamoto M, Saijoh Y, Perea-Gomez A, Shawlot W, Behringer RR, Ang SL, Hamada H, Meno C (2004) Nodal antagonists regulate formation of the anteroposterior axis of the mouse embryo. Nature 428:387–392.CrossRefGoogle Scholar

Copyright information

© Springer 2009

Authors and Affiliations

  1. 1.Department of PhysiologyDevelopment and Neuroscience, University of CambridgeCambridgeUK

Personalised recommendations