Transporting Mouse Embryos and Germplasm as Frozen or Unfrozen Materials

Janet Kenyon1, Mo Guan1, Debora Bogani1, Susan Marschall2, Marcello Raspa3, Amanda Pickard4, Toru Takeo5, Naomi Nakagata5, Martin Fray1

1 Mary Lyon Centre, Medical Research Council, Harwell Science and Innovation Campus, Oxfordshire, 2 Institute of Experimental Genetics, Helmholtz Zentrum München—German Research Center for Environmental Health (GmbH), Neuherberg, 3 Consiglio Nazionale delle Ricerche (IBCN) CNR‐Campus International Development (EMMA‐INFRAFRONTIER‐IMPC), A. Buzzati‐Traverso Campus, Rome, 4 Division of Biomedical Services, University of Leicester, Leicester, 5 University of Kumamoto, Kumamoto
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo140064
Online Posting Date:  June, 2014
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Abstract

The 21st century has seen a huge proliferation in the availability of genetically altered mice. The availability of these resources has been accompanied by ever greater opportunities for international collaborations between laboratories involving the exchange of mouse strains. This exchange can involve significant costs in terms of animal welfare and transportation expenses. In an attempt to mitigate some of these costs, the mouse community has developed a battery of techniques that can be used to avoid transporting live mice. Transporting frozen embryos and sperm at liquid nitrogen (LN2) temperatures using dry shippers has been common practice for some time. However, current advances in this field have refined transportation procedures and introduced new techniques for disseminating embryos and sperm: for example, shipping frozen sperm on dry ice, exchanging unfrozen epididymides from which sperm can be extracted, and transporting frozen/thawed embryos in isotonic media. This article discusses some of the current practices used by laboratories to transport mouse strains around the world without having to exchange live mice. Curr. Protoc. Mouse Biol. 4:47‐65 © 2014 by John Wiley & Sons, Inc.

Keywords: cryopreservation; mouse; embryos; spermatozoa; epididymides; transport

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Transporting Frozen Materials Using LN2 Dry Shippers
  • Basic Protocol 2: Transporting Frozen Sperm on Dry Ice
  • Basic Protocol 3: Transporting Unfrozen Epididymides
  • Basic Protocol 4: Transporting Frozen/Thawed Embryos
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Transporting Frozen Materials Using LN2 Dry Shippers

  Materials
  • Liquid nitrogen
  • Frozen samples to be transported (see protocols below)
  • Dewar suitable for carrying LN 2
  • Tipping Dewar suitable for pouring LN 2
  • Dry shippers (e.g., Taylor Wharton)
  • Plastic goblet (e.g., Planer, cat. no. FZPA‐005, white)
  • Cryo‐canes (e.g., Scientific Laboratory Supplies, cat. no. 378441) and cryotubes (e.g., VWR, cat. no. 479‐6837)
NOTE: Before filling the dry shipper make sure that you are wearing the appropriate personal protective equipment, which should include insulated gloves, an apron, and eye protection

Basic Protocol 2: Transporting Frozen Sperm on Dry Ice

  Materials
  • Sperm samples frozen in sperm straws using conventional techniques
  • Dry ice
  • Insulating polystyrene box to prepare dry‐ice parcel
  • Cassette for holding sperm straws (e.g., Hunter Scientific, cat. no. 16980/0601)

Basic Protocol 3: Transporting Unfrozen Epididymides

  Materials
  • Mature male mice over 8 weeks of age
  • LiFor preservation medium (Lifeblood Medical Inc.) or mineral oil (Sigma, cat. no. M8410)
  • Sphingosine‐1‐phosphate (S1P; Cambridge Bioscience, cat. no. S6130)
  • 70% ethanol
  • 0.2‐ml microcentrifuge tube (e.g., Fisher, cat. no. TUP‐114‐010Q)
  • Dissecting instruments e.g., standard dissecting forceps, fine watchmakers forceps, and scissors
  • 7‐ml plastic bijou vial (Sigma, cat. no. Z645338)
  • Ice packs (e.g., Fisher, cat. no. ICE‐910‐020W)
  • Polyethylene bags
  • 0.5‐liter thermos flask (e.g., ThermoCafé)
  • Optional: Maxim Integrated Products iButton Thermochron F5 (Digi‐Key Corporation, cat. no. DS1921G‐F5#‐ND)
  • Biotransporter boxes (e.g., Air Sea Containers, Code 570)
  • Packing tape
  • Dissecting microscope
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, )

Basic Protocol 4: Transporting Frozen/Thawed Embryos

  Materials
  • Frozen/thawed embryos (2‐cell, morulae, or blastocysts)
  • KSOM medium (e.g., Millipore/Chemicon, cat. no. MR‐020P‐5F)
  • M2 medium (e.g., Sigma, cat. no. M7167)
  • 60‐mm culture dishes (e.g., Falcon: 351016)
  • Aspirator assembly (Sigma, cat. no. A5177)
  • 0.5‐ml microcentrifuge tube (e.g., Fisher, cat. no. TUP‐952‐010W)
  • 7.0‐ml plastic bijou vial (Sigma, cat. no. Z645338)
  • Ice packs (e.g., Fisher, cat. no. ICE‐910‐020W)
  • Polyethylene bags
  • 0.5‐liter thermos flask (e.g., ThermoCafé)
  • Optional: Maxim Integrated Products iButton Thermochron F5 (Digi‐Key Corporation, cat. no. DS1921G‐F5#‐ND)
  • Biotransporter boxes (e.g., Air Sea Containers, Code 570)
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Figures

Videos

Literature Cited

Literature Cited
  Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
  Mochida, K., Ohkawa, M., Inoue, K., Valdez, D.M., Jr., Kasai, M., and Ogura, A. 2005. Birth of mice after in vitro fertilization using C57BL/6 sperm transported within epididymides at refrigerated temperatures. Theriogenology 64:135‐143.
  Mochida, K., Hasegawa, A., Li, M.W., Fray, M.D., Kito, S., Vallelunga, J.M., Lloyd, K.C., Yoshiki, A., Obata, Y., and Ogura, A. 2013. High osmolality vitrification: A new method for the simple and temperature‐permissive cryopreservation of mouse embryos. PloS One 8:e49316.
  Okamoto, M., Nakagata, N., and Toyoda, Y. 2001. Cryopreservation and transport of mouse spermatozoa at −79°C. Exp. Anim. 50:83‐86.
  Ostermeier, G.C., Wiles, M.V., Farley, J.S., and Taft, R.A. 2008. Conserving, distributing and managing genetically modified mouse lines by sperm cryopreservation. PLoS One 3:e2792.
  Takeo, T., and Nakagata, N. 2011. Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl‐beta‐cyclodextrin. Biol Reprod 85:1066‐1072.
  Takeo, T., Kaneko, T., Haruguchi, Y., Fukumoto, K., Machida, H., Koga, M., Nakagawa, Y., Takeshita, Y., Matsuguma, T., Tsuchiyama, S., Shimizu, N., Hasegawa, T., Goto, M., Miyachi, H., Anzai, M., Nakatsukasa, E., Nomaru, K., and Nakagata, N. 2009. Birth of mice from vitrified/warmed 2‐cell embryos transported at a cold temperature. Cryobiology 58:196‐202.
  Takeo, T., Kondo, T., Haruguchi, Y., Fukumoto, K., Nakagawa, Y., Takeshita, Y., Nakamuta, Y., Tsuchiyama, S., Shimizu, N., Hasegawa, T., Goto, M., Miyachi, H., Anzai, M., Fujikawa, R., Nomaru, K., Kaneko, T., Itagaki, Y., and Nakagata, N. 2010. Short‐term storage and transport at cold temperatures of 2‐cell mouse embryos produced by cryopreserved sperm. J. Am. Assoc. Lab. Anim. Sci. 49:415‐419.
  Takeo, T., Tsutsumi, A., Omaru, T., Fukumoto, K., Haruguchi, Y., Kondo, T., Nakamuta, Y., Takeshita, Y., Matsunaga, H., Tsuchiyama, S., Sakoh, K., Nakao, S., Yoshimoto, H., Shimizu, N., and Nakagata, N. 2012. Establishment of a transport system for mouse epididymal sperm at refrigerated temperatures. Cryobiology 65:163‐168.
  Takeo, T., Fukumoto, K., Kondo, T., Haruguchi, Y., Takeshita, Y., Nakamuta, Y., Tsuchiyama, S., Yoshimoto, H., Shimizu, N., Li, M.W., Kinchen, K., Vallelunga, J., Kent Lloyd, K.C., and Nakagata, N. 2013. Investigations of motility and fertilization potential in thawed cryopreserved mouse sperm from cold‐stored epididymides. Cryobiology 68:12‐17.
Internet Resources
  http://www.infrafrontier.eu
  The European Mouse Mutant Archive (Europe), which hosts numerous Web‐based source materials of use to cryobiologists
  http://card.medic.kumamoto‐u.ac.jp/card/english/index.html
  The Center for Animal Resources and Development (Japan), which hosts numerous Web‐based source materials of use to cryobiologists
  http://www.brc.riken.jp/inf/en/index.shtml
  The RIKEN BioResource Center, which hosts numerous Web‐based source materials of use to cryobiologists
  http://jax.org
  The Jackson Laboratory (USA), which hosts numerous Web‐based source materials of use to cryobiologists
  https://www.mmrrc.org/
  The Web site for Mutant Mouse Regional Resource Centre (USA), which hosts numerous Web‐based source materials of use to cryobiologists
  http://findmice.org
  A searchable Web site for the International Mouse Strain Resource (IMSR), which links to mouse resources around the world.
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