Serum-Free Culture of Mid-gestation Mouse Embryos: A Tool for the Study of Endoderm-Derived Organs

  • Julie Gordon
  • Billie A. Moore
  • C. Clare Blackburn
  • Nancy R. Manley
Part of the Methods in Molecular Biology book series (MIMB, volume 1092)


The experimental manipulation of mid-gestation mouse embryos is an important tool for the study of developmental biology. However, such techniques can be challenging due to difficulties accessing the embryos in utero, and therefore the ability to maintain mid-gestation mouse embryos in vitro has proved invaluable. Described here is an example of a whole embryo culture system, where a serum-free medium is used to support the development of mouse embryos in vitro from embryonic day 10.5 (E10.5) to E11.5. During this time the embryos increase in size and undergo developmental progression, as determined by morphological and molecular criteria. This makes it an ideal environment in which to support and maintain mid-gestation mouse embryos following experimental manipulations.

Two applications of this whole embryo culture system are described here. In the first, protein-soaked beads are carefully positioned in the pharyngeal region of an E10.5 embryo, allowing the concentration of specific proteins to be altered within the tissue. In the second technique, morpholino oligonucleotides are electroporated into the pharyngeal region of the embryo at E10.5, creating an efficient system for the knockdown of gene function in the target cells. These techniques demonstrate the use of in vitro techniques to study organogenesis within the pharyngeal region of the mouse embryo, but with some modification they could be adapted to target any region of the endodermal gut tube.

Key words

Serum-free whole embryo culture Mouse embryogenesis Bead implants Electroporation Morpholinos Endoderm 



This work was supported by LRF and MRC (for J.G. and C.C.B.) and by NIH, NICHD and NIAID (for N.R.M. and B.M.).


  1. 1.
    New DAT (1990) Whole embryo culture, teratogenesis and the estimation of teratologic risk. Teratology 42:635–642PubMedCrossRefGoogle Scholar
  2. 2.
    New DAT, Coppola PT, Cockroft DL (1976) J Embryol Exp Morphol 36:133–144PubMedGoogle Scholar
  3. 3.
    Quinn P, Horstman FC (1998) Is the mouse a good model for the human with respect to the development of the preimplantation embryo in vitro? Hum Reprod 13:173–183PubMedCrossRefGoogle Scholar
  4. 4.
    Kinder SJ, Tan S-S, Tam PPL (2000) Cell grafting and cell fate mapping of the early-somite stage mouse embryo. Methods Mol Biol Dev Biol Protocols 135:425–437CrossRefGoogle Scholar
  5. 5.
    Tam PPL (1998) Postimplantation mouse development: whole embryo culture and micro-manipulation. Int J Dev Biol 42:895–902PubMedGoogle Scholar
  6. 6.
    Takahashi M, Osumi N (2010) The method of rodent whole embryo culture using the rotator-type bottle culture system. J Vis Exp 42.
  7. 7.
    Moore-Scott BA, Gordon J, Blackburn CC, Condie BG, Manley NR (2003) New serum-free in vitro culture technique for midgestation mouse embryos. Genesis 35:164–168PubMedCrossRefGoogle Scholar
  8. 8.
    Gordon J, Wilson VA, Blair NF, Sharp L, Manley NR, Blackburn CC (2002) Functional evidence for a single endodermal origin for the thymic epithelium. Nat Immunol 5:546–553CrossRefGoogle Scholar
  9. 9.
    Cockroft DL (1991) Culture media for postimplantation embryos. Reprod Toxicol 5:223–228PubMedCrossRefGoogle Scholar
  10. 10.
    Van Maele-Fabry G, Picard JJ, Attenon P, Berthet P, Delhaise F, Govers MJAP, Peters PWJ, Piersma AH, Schmid BP, Stadler J, Verhoef A, Verseil C (1991) Interlaboratory evaluation of three culture media for postimplantation rodent embryos. Reprod Toxicol 5:417–426PubMedCrossRefGoogle Scholar
  11. 11.
    Akamatsu W, Okano HJ, Osumi N, Inoue T, Nakamura S, Sakakibara S-I, Miura M, Matsuo N, Darnell RB, Okano H (1999) Mammalian ELAV-like neuronal RNA-binding proteins HuB and HuC promote neuronal development in both the central and the peripheral nervous system. Proc Natl Acad Sci U S A 96:9885–9890PubMedCrossRefGoogle Scholar
  12. 12.
    Osumi N, Inoue T (2001) Gene transfer into cultured mammalian embryos by electroporation. Methods 24:35–42PubMedCrossRefGoogle Scholar
  13. 13.
    Sadler TW, New DAT (1981) Culture of mouse embryos during neurulation. J Embryol Exp Morphol 66:109–116PubMedGoogle Scholar
  14. 14.
    Heasman J (2002) Morpholino oligonucleotides: making sense of antisense? Dev Biol 243:209–214PubMedCrossRefGoogle Scholar
  15. 15.
    Nasevicius A, Ekker SC (2000) Effective targeted gene “knockdown” in zebrafish. Nat Genet 26:216–220PubMedCrossRefGoogle Scholar
  16. 16.
    Coonrod SA, Bolling LC, Wright PW, Visconti PE, Herr JC (2001) A morpholino phenocopy of the mouse mos mutation. Genesis 30:198–200PubMedCrossRefGoogle Scholar
  17. 17.
    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
  18. 18.
    Garcia-Frigola C, Carreres MI, Vegar C, Herrera E (2007) Gene delivery into mouse retinal ganglion cells by in utero electroporation. BMC Dev Biol 7:103–112PubMedCrossRefGoogle Scholar
  19. 19.
    Saito T (2006) In vivo electroporation in the embryonic mouse central nervous system. Nat Protoc 1:1552–1558PubMedCrossRefGoogle Scholar
  20. 20.
    Akamatsu W, Okano HJ, Osumi N, Inoue T, Nakamura S, Sakakibara S, Miura M, Matsuo N, Darnell RB, Okano H (1999) Mammalian ELAV-like neuronal RNA-binding proteins HuB and HuC promote neuronal development in both the central and the peripheral nervous systems. PNAS 96:9885–9890PubMedCrossRefGoogle Scholar
  21. 21.
    Kaufman MH (1992) The atlas of mouse development. Academic, LondonGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Julie Gordon
    • 1
  • Billie A. Moore
    • 1
  • C. Clare Blackburn
    • 2
  • Nancy R. Manley
    • 1
  1. 1.Department of GeneticsUniversity of GeorgiaAthensUSA
  2. 2.MRC Centre for Regenerative Medicine, Institute for Stem Cell ResearchUniversity of EdinburghEdinburghUK

Personalised recommendations