Preparation of rat serum suitable for mammalian whole embryo culture.

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

[1]  J. Lauderdale,et al.  Mouse embryonic development in a serum-free whole embryo culture system. , 2014, Journal of visualized experiments : JoVE.

[2]  M. Kyba,et al.  Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons , 2014, Development.

[3]  T. Obayashi,et al.  Dmrta1 regulates proneural gene expression downstream of Pax6 in the mammalian telencephalon , 2013, Genes to cells : devoted to molecular & cellular mechanisms.

[4]  R. Arkell,et al.  Successful whole embryo culture with commercially available reagents. , 2013, The International journal of developmental biology.

[5]  M. Ross,et al.  Neural Tube Closure in Mouse Whole Embryo Culture , 2011, Journal of visualized experiments : JoVE.

[6]  Ryan S. Udan,et al.  Preparation of rat serum for culturing mouse embryos. , 2011, Cold Spring Harbor protocols.

[7]  T. Pierfelice,et al.  Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5 , 2010, Journal of visualized experiments : JoVE.

[8]  N. Osumi,et al.  The method of rodent whole embryo culture using the rotator-type bottle culture system. , 2010, Journal of visualized experiments : JoVE.

[9]  Noriko Osumi,et al.  Transferring genes into cultured mammalian embryos by electroporation , 2008, Development, growth & differentiation.

[10]  R. Behringer,et al.  Roller culture of postimplantation embryos. , 2006, CSH protocols.

[11]  J. Frisén,et al.  Pax6-dependent boundary defines alignment of migrating olfactory cortex neurons via the repulsive activity of ephrin A5 , 2006, Development.

[12]  P. Brown-Woodman,et al.  Replacement of ether with alternate volatile anesthetics for collection of rat serum used in embryo culture. , 2004, Toxicology in vitro : an international journal published in association with BIBRA.

[13]  Y. Saijoh,et al.  Nodal antagonists regulate formation of the anteroposterior axis of the mouse embryo , 2004, Nature.

[14]  A. Ornoy,et al.  A simple method of culture of 11.5‐day‐old rat embryos in DMEM/F12 and 20% fetal bovine serum , 2003, Journal of anatomy.

[15]  N. Manley,et al.  New serum‐free in vitro culture technique for midgestation mouse embryos , 2003, Genesis.

[16]  W. Huttner,et al.  Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[17]  N. Osumi,et al.  Pax6 regulates specification of ventral neurone subtypes in the hindbrain by establishing progenitor domains. , 2002, Development.

[18]  N. Nakatsuji,et al.  Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. , 2001, Developmental biology.

[19]  E. Grove,et al.  Neocortex Patterning by the Secreted Signaling Molecule FGF8 , 2001, Science.

[20]  H. Tabata,et al.  Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex , 2001, Neuroscience.

[21]  M. Takeichi,et al.  Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development. , 2001, Development.

[22]  T. Matsuo,et al.  A mutation in the Pax-6 gene in rat small eye is associated with impaired migration of midbrain crest cells , 1993, Nature Genetics.

[23]  D. New.  Whole embryo culture, teratogenesis, and the estimation of teratologic risk. , 1990, Teratology.

[24]  N. Greene,et al.  Convergent extension analysis in mouse whole embryo culture. , 2012, Methods in molecular biology.

[25]  P. Khoo,et al.  Cell grafting and labeling in postimplantation mouse embryos. , 2008, Methods in molecular biology.

[26]  D. Turnbull,et al.  Ultrasound backscatter microscopy of mouse embryos. , 2000, Methods in molecular biology.

[27]  B. Hogan,et al.  Manipulating the mouse embryo: A laboratory manual , 1986 .

[28]  K. Eto,et al.  Improved development of rat embryos in culture during the period of craniofacial morphogenesis. , 1985, Journal of craniofacial genetics and developmental biology.