Conservation of Mouse Models Through Embryo Freezing

Mo Guan1, Debora Bogani1, Susan Marschall2, Marcello Raspa3, Toru Takeo4, Naomi Nakagata4, Martin Fray1

1 Mary Lyon Centre, Medical Research Council, Oxfordshire, 2 Institute of Experimental Genetics, Helmholtz Zentrum Muenchen ‐ German Research Center for Environmental Health (GmbH), Neuherberg, 3 Consiglio Nazionale delle Ricerche (IBCN) CNR‐Campus International Development (EMMA‐INFRAFRONTIER‐IMPC), Rome, 4 Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo140082
Online Posting Date:  December, 2014
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Abstract

The ability to interrogate the entire coding sequence of the mouse combined with the tools to manipulate the genome has firmly established the mouse as the model organism of choice for studying the causes of human disease. Consequently, a huge number of novel mouse models are generated each year to support active research programs. However, it is neither ethically justifiable, nor economically viable to maintain mouse colonies on the shelf that are not part of active research programs. This means that novel mouse lines have to be preserved in some way. If this is not done and the line is simply killed off, the genetics will be lost to future generations of scientists. This article describes the current practices used in cryopreservation laboratories to archive and recover mouse embryos frozen using controlled‐rate freezing and vitrification techniques. © 2014 by John Wiley & Sons, Inc.

Keywords: cryopreservation; mouse; pre‐implantation; embryos; vitrification

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Controlled‐Rate Freezing of Pre‐Implantation Mouse Embryos
  • Alternate Protocol 1: A Robust Vitrification Method for Cryopreserving Mouse Embryos
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Controlled‐Rate Freezing of Pre‐Implantation Mouse Embryos

  Materials
  • 3‐ to 4‐week‐old female mice (e.g., weight and strain will depend on the stock being frozen)
  • Pregnant mare's serum gonadotrophin (e.g., Harbor UCLA R.E.I. National Hor, NHPP‐3383A)
  • Human chorionic gonadotrophin (e.g., Intervet)
  • Mature males of proven fertility
  • 70% (v/v) ethanol
  • M2 medium (Sigma, cat. no. M7167)
  • Propylene glycol (ProH; see recipe)
  • Sucrose solution (see recipe)
  • Heat sealer or capillary sealant (e.g., Cristaseal)
  • Liquid nitrogen (LN 2)
  • KSOM (e.g., Millipore/Chemicon, cat. no. MR‐020P‐5F)
  • MEM amino acid solution (50×) (e.g., Life Technologies, cat. no. 11130036)
  • MEM non‐essential amino acid solution (100×) (e.g., Sigma, cat. no. M7145‐100ML)
  • 200‐μl and 1‐ml pipets, plus tips (e.g., Gilson)
  • 1‐ml syringe equipped with a 25‐G needle
  • Dissecting instruments: e.g., standard dissecting forceps, fine watchmakers forceps, and scissors
  • 60‐mm culture dishes (e.g., Falcon, cat. no. 351016)
  • 1‐ml syringes
  • 30‐ and 33‐G flushing needles (e.g., Coppers)
  • Dissecting microscope (10×‐100× magnification)
  • 35‐mm culture dishes (e.g., Falcon, cat. no. 351008)
  • Mouth pipet aspirator assembly (e.g., Sigma, cat. no. A5177)
  • Capillary pipets
  • Controlled‐rate freezing machine (e.g., Planar or Biocool)
  • 0.25 ml French straws (e.g., Planer, cat. no. FZA201)
  • 16 g metal rod (∼18-cm long)
  • Indelible marker pen or cryo‐fast labels (e.g., Brady labels)
  • Laboratory timers
  • Cotton wool buds
  • Small dewar suitable for holding LN 2
  • Water bath at room temperature (∼20°C)
  • Paper tissues
  • Additional reagents and equipment for euthanizing the mice (Donovan and Brown, )

Alternate Protocol 1: A Robust Vitrification Method for Cryopreserving Mouse Embryos

  Materials
  • Crush ice
  • Water
  • DAP213 vitrification solution (see recipe)
  • 1 M dimethyl sulfoxide (DMSO) solution in PB1
  • PB1 solution (see recipe)
  • Liquid nitrogen (LN 2)
  • 0.25 M sucrose in PB1 as warming solution (see recipe)
  • KSOM (e.g., Millipore/Chemicon, cat. no. MR‐020P‐5F)
  • MEM amino acid solution (50×) (e.g., Life Technologies, cat. no. 11130036)
  • MEM non‐essential amino acid solution (100×) (e.g., Sigma, cat. no. M7145‐100ML)
  • 20‐μl, 200‐μl, and 1‐ml pipets and tips (e.g., Gilson)
  • 0.22‐μm syringe end filters
  • Mr. Frosty freezing container (Nalgene, cat. no. 5100‐001)
  • Cryovials (Sumitomo Bakelite, cat. no. MS‐4501W;)
  • 60‐mm culture dishes (Falcon, cat. no. 351016)
  • Embryo‐handling pipets
  • Capillary pipets
  • Mouth pipet/aspirator assembly (e.g., Sigma, cat. no. A5177)
  • Aluminum canes for supporting cryovials (SLS, cat. no. 378441)
  • Small dewar for holding LN 2
  • 35‐mm culture dishes (Falcon, cat. no. 351008)
  • 37°C, 5% CO 2 incubator
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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.
  Glenister, P.H. and Lyon, M.F. 1986. Long‐term storage of eight‐cell mouse embryos at −196 degrees C. J. In Vitro Fertil. Embryo Transfer 3:20‐27.
  Karran, G. and Legge, M. 1996. Non‐enzymatic formation of formaldehyde in mouse oocyte freezing mixtures. Hum. Reprod. 11:2681‐2686.
  Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. 2003. Manipulating the Mouse Embryo—A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, New York.
  Nakao, K., Nakagata, N., and Katsuki, M. 1997. Simple and efficient vitrification procedure for cryopreservation of mouse embryos. Exp. Anim. 46:231‐234.
  Rall, W.F. and Fahy, G.M. 1985. Ice‐free cryopreservation of mouse embryos at −196 degrees C by vitrification. Nature 313:573‐575.
  Renard, J.P. and Babinet, C. 1984. High survival of mouse embryos after rapid freezing and thawing inside plastic straws with 1‐2 propanediol as cryoprotectant. J. Exp. Zool. 230:443‐448.
  Scavizzi, F. and Raspa, M. 2006. Helicobacter typhlonius was detected in the sex organs of three mouse strains but did not transmit vertically. Lab. Anim. 40:70‐79.
  Stringfellow, D.A. and Seidel, S. 1998. Manual of the International Embryo Transfer Society, 3rd Ed. IETS, Savoy, Illinois.
  Whittingham, D.G., Leibo, S.P., and Mazur, P. 1972. Survival of mouse embryos frozen to −196 degrees and −269°C. Science 178:411‐414.
  Wilmut, I. 1972. The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing. Life Sci. II 11:1071‐1079.
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 website for Mutant Mouse Regional Resource Center (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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