Analyzing Gene Function in Whole Mouse Embryo and Fetal Organ In Vitro

  • Satomi S. Tanaka
  • Yasuka L. Yamaguchi
  • Vanessa J. Jones
  • Patrick P. L. Tam
Part of the Methods in Molecular Biology book series (MIMB, volume 1092)


A well-established experimental paradigm to analyze gene function in development is to elucidate the impact of gain and loss of gene activity on cell differentiation, tissue modelling, organogenesis, and morphogenesis. This chapter describes the experimental protocols to study gene function by means of electroporation and lipofection to manipulate genetic activity in whole embryos and fetal organs in vitro. These techniques allow for more precise control of the timing, with reference to developmental age or stage, and the cell/tissue-specificity of the changes in gene activity. They provide an alternative strategy that can expedite the analysis of gene function before resorting to the conventional means of transgenesis and gene targeting in the whole organism.

Key words

Mouse embryo Fetal ovary In vitro culture Electroporation Lipofection siRNA 


  1. 1.
    Tam PP, Khoo PL, Lewis SL, Bildsoe H, Wong N, Tsang TE, Gad JM, Robb L (2007) Sequential allocation and global pattern of movement of the definitive endoderm in the mouse embryo during gastrulation. Development 134:251–260PubMedCrossRefGoogle Scholar
  2. 2.
    Uchikawa M, Ishida Y, Takemoto T, Kamachi Y, Kondoh H (2003) Functional analysis of chicken Sox2 enhancers highlights an array of diverse regulatory elements that are conserved in mammals. Dev Cell 4:509–519PubMedCrossRefGoogle Scholar
  3. 3.
    Davidson BP, Tsang TE, Khoo PL, Gad JM, Tam PP (2003) Introduction of cell markers into germ layer tissues of the mouse gastrula by whole embryo electroporation. Genesis 35:57–62PubMedCrossRefGoogle Scholar
  4. 4.
    Tanaka SS, Yamaguchi YL, Tsoi B, Lickert H, Tam PP (2005) IFITM/Mil/fragilis family proteins IFITM1 and IFITM3 play distinct roles in mouse primordial germ cell homing and repulsion. Dev Cell 9:745–756PubMedCrossRefGoogle Scholar
  5. 5.
    Ogura T (2002) In vivo electroporation: a new frontier for gene delivery and embryology. Differentiation 70:163–171PubMedCrossRefGoogle Scholar
  6. 6.
    Lickert H, Cox B, Wehrle C, Taketo MM, Kemler R, Rossant J (2005) Dissecting Wnt/beta-catenin signaling during gastrulation using RNA interference in mouse embryos. Development 132:2599–2609PubMedCrossRefGoogle Scholar
  7. 7.
    Itasaki N, Bel-Vialar S, Krumlauf R (1999) ‘Shocking’ developments in chick embryology: electroporation and in ovo gene expression. Nat Cell Biol 1:E203–E207PubMedCrossRefGoogle Scholar
  8. 8.
    Swartz M, Eberhart J, Mastick GS, Krull CE (2001) Sparking new frontiers: using in vivo electroporation for genetic manipulations. Dev Biol 233:13–21PubMedCrossRefGoogle Scholar
  9. 9.
    Saito T, Nakatsuji N (2001) Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev Biol 240:237–246PubMedCrossRefGoogle Scholar
  10. 10.
    Tabata H, Nakajima K (2001) Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex. Neuroscience 103:865–872PubMedCrossRefGoogle Scholar
  11. 11.
    Fukuchi-Shimogori T, Grove EA (2001) Neocortex patterning by the secreted signaling molecule FGF8. Science 294:1071–1074PubMedCrossRefGoogle Scholar
  12. 12.
    Fukuchi-Shimogori T, Grove EA (2003) Emx2 patterns the neocortex by regulating FGF positional signaling. Nat Neurosci 6:825–831PubMedCrossRefGoogle Scholar
  13. 13.
    Osumi N, Inoue T (2001) Gene transfer into cultured mammalian embryos by electroporation. Methods 24:35–42PubMedCrossRefGoogle Scholar
  14. 14.
    Takahashi M, Sato K, Nomura T, Osumi N (2002) Manipulating gene expressions by electroporation in the developing brain of mammalian embryos. Differentiation 70:155–162PubMedCrossRefGoogle Scholar
  15. 15.
    deCastro M, Saijoh Y, Schoenwolf GC (2006) Optimized cationic lipid-based gene delivery reagents for use in developing vertebrate embryos. Dev Dyn 235:2210–2219PubMedCrossRefGoogle Scholar
  16. 16.
    Yamaguchi YL, Tanaka SS, Yasuda K, Matsui Y, Tam PP (2006) Stage-specific Importin13 activity influences meiosis of germ cells in the mouse. Dev Biol 297:350–360PubMedCrossRefGoogle Scholar
  17. 17.
    Tam PP (1998) Postimplantation mouse development: whole embryo culture and micro-manipulation. Int J Dev Biol 42:895–902PubMedGoogle Scholar
  18. 18.
    Franklin V, Bildsoe H, Tam PP (2007) Fate-mapping technique: grafting fluorescent cells into gastrula stage mouse embryos at 7–7.5 days post coitum. CSH Protoc. doi:10.1101/pdb.prot4892Google Scholar
  19. 19.
    Khoo PL, Franklin VJ, Tam PP (2007) Fate-mapping technique: targeted whole embryo electroporation of DNA constructs into the germ layers of 7–7.5 dpc mouse embryos. CSH Protoc. doi: 10.1101/pdb.prot4893
  20. 20.
    Nagy A, Gertenstein M, Vintersten K, Behringer R (2003) Isolation, culture and manipulation of postimplantation embryos, Chapter 5 “Manipulating the Mouse Embryo”, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  21. 21.
    Donovan SL, Dyer MA (2006) Preparation and square wave electroporation of retinal explant cultures. Nat Protoc 1:2710–2718PubMedCrossRefGoogle Scholar
  22. 22.
    Alie TM, Vrljicak PJ, Myburgh DB, Gupta IR (2007) Microinjection and electroporation of embryonic kidney explants: an improved method. Kidney Int 72:121–125PubMedCrossRefGoogle Scholar
  23. 23.
    Lee JM, Kim JY, Cho KW, Lee MJ, Cho SW, Kwak S, Cai J, Jung HS (2008) Wnt11/Fgfr1b cross-talk modulates the fate of cells in palate development. Dev Biol 314:341–350PubMedCrossRefGoogle Scholar
  24. 24.
    Hiramatsu R, Kanai Y, Mizukami T, Ishii M, Matoba S, Kanai-Azuma M, Kurohmaru M, Kawakami H, Hayashi Y (2003) Regionally distinct potencies of mouse XY genital ridge to initiate testis differentiation dependent on anteroposterior axis. Dev Dyn 228:247–253PubMedCrossRefGoogle Scholar
  25. 25.
    Davies JA, Ladomery M, Hohenstein P, Michael L, Shafe A, Spraggon L, Hastie N (2004) Development of an siRNA-based method for repressing specific genes in renal organ culture and its use to show that the Wt1 tumour suppressor is required for nephron differentiation. Hum Mol Genet 13:235–246PubMedCrossRefGoogle Scholar
  26. 26.
    Liu J, Beqaj S, Yang Y, Honore B, Schuger L (2001) Heterogeneous nuclear ribonucleoprotein-H plays a suppressive role in visceral myogenesis. Mech Dev 104:79–87PubMedCrossRefGoogle Scholar
  27. 27.
    Nishinakamura R, Matsumoto Y, Nakao K, Nakamura K, Sato A, Copeland NG, Gilbert DJ, Jenkins NA, Scully S, Lacey DL, Katsuki M, Asashima M, Yokota T (2001) Murine homolog of SALL1 is essential for ureteric bud invasion in kidney development. Development 128:3105–3115PubMedGoogle Scholar
  28. 28.
    Park EH, Taketo T (2003) Onset and progress of meiotic prophase in the oocytes in the B6.YTIR sex-reversed mouse ovary. Biol Reprod 69:1879–1889PubMedCrossRefGoogle Scholar
  29. 29.
    Okada Y, Ueshin Y, Isotani A, Saito-Fujita T, Nakashima H, Kimura K, Mizoguchi A, Oh-Hora M, Mori Y, Ogata M, Oshima RG, Okabe M, Ikawa M (2007) Complementation of placental defects and embryonic lethality by trophoblast-specific lentiviral gene transfer. Nat Biotechnol 25:233–237PubMedCrossRefGoogle Scholar
  30. 30.
    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 postimplantation mouse embryos. Mech Dev 118:57–63PubMedCrossRefGoogle Scholar
  31. 31.
    Bartkowska K, Paquin A, Gauthier AS, Kaplan DR, Miller FD (2007) Trk signaling regulates neural precursor cell proliferation and differentiation during cortical development. Development 134:4369–4380PubMedCrossRefGoogle Scholar
  32. 32.
    Calegari F, Haubensak W, Yang D, Huttner WB, Buchholz F (2002) Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA. Proc Natl Acad Sci U S A 99:14236–14240PubMedCrossRefGoogle Scholar
  33. 33.
    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 428:387–392PubMedCrossRefGoogle Scholar
  34. 34.
    Tanaka SS, Yamaguchi YL, Steiner KA, Nakano T, Nishinakamura R, Kwan KM, Behringer RR, Tam PP (2010) Loss of Lhx1 activity impacts on the localization of primordial germ cells in the mouse. Dev Dyn 239:2851–2859PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Satomi S. Tanaka
    • 1
  • Yasuka L. Yamaguchi
    • 1
  • Vanessa J. Jones
    • 2
  • Patrick P. L. Tam
    • 2
  1. 1.Department of Kidney Development, Institute of Molecular Embryology and GeneticsKumamoto UniversityKumamotoJapan
  2. 2.Children’s Medical Research Institute, Sydney Medical SchoolThe University of SydneySydneyAustralia

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