Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 May;1(5):e138.
doi: 10.1002/cpz1.138.

Cryopreservation Protocols for Genetically Engineered Mice

Glenn Longenecker  1 , Kyoungin Cho  2 , Jaspal S Khillan  2 , Ashok B Kulkarni  1
Affiliations

Cryopreservation Protocols for Genetically Engineered Mice

Glenn Longenecker et al. Curr Protoc. 2021 May.

Erratum in

Abstract

Protocols for cryopreservation of mouse embryos and sperm are important for preserving genetically engineered mice (GEMs) used in research to study human development and diseases. Embryo cryopreservation is mainly carried out using either of two protocols: controlled gradual cooling or vitrification. Sperm cryopreservation protocols include two methodologies that are commonly referred to as JAX and CARD. Quality-control measures are necessary to ensure that GEMs are properly cryopreserved so that they can be retrieved for future use. An archiving system is also important in keeping proper records of frozen sperm and embryos. Frozen embryos and sperm are now preferred over live mice for shipping to distant locations. This article describes detailed protocols used in cryopreservation of mouse embryos and sperm, as well as their retrieval to live mice. © 2021 U.S. Government. Sperm cryopreservation Basic Protocol 1: JAX protocol for sperm cryopreservation Support Protocol 1: JAX protocol for making sperm cryopreservation medium Basic Protocol 2: JAX protocol for IVF of mouse sperm Alternate Protocol 1: Modified CARD protocol for sperm cryopreservation Support Protocol 2: CARD protocol for making sperm cryopreservation medium Alternate Protocol 2: CARD protocol for IVF of mouse sperm Embryo cryopreservation Basic Protocol 3: Cryopreserving and thawing 2-cell embryos Alternate Protocol 3: Cryopreserving and thawing 8-cell to morula-stage embryos Surgical transfer of embryos Basic Protocol 4: Infundibulum transfer of 2-cell to morula-stage embryos.

Keywords: In Vitro Fertilization (IVF); controlled rate freezing; embryo cryopreservation; sperm cryopreservation; vitrification.

PubMed Disclaimer

Conflict of interest statement

Conflict of Interest

None

Figures

Figure 1.
Figure 1.
Sperm cryopreservation area. This is the area where the mouse sperm is harvested, collected, loaded into straws, and frozen down. From left to right that includes a dissecting microscope, light source, humidification chamber, straw sealer, water bath, and the gray freezing container.
Figure 2.
Figure 2.
Loading of sperm cryopreservation straws. (A) Unloaded and marked straw. Mark 1 is the for the uptake of the 10 mm of CPM sperm media suspension that will seal the top plug end of the straw. Mark 2 is for the uptake of the 10 mm of CPM/sperm suspension prior to the addition of 5 mm sperm suspension aliquots. Mark 3 is for the 5 mm CPM/sperm suspension aliquots to be used for IVF. (B) Marked and loaded straw. (C) Loaded and sealed straw.
Figure 3.
Figure 3.
IVF and embryo surgical transfer area. This is the area where the oocytes are harvested and collected, and the mouse sperm is added to the IVF plates. It is also the same area where the embryo transfers are done. From left to right this includes the COOK IVF incubator, dissecting microscope for surgical transfer of embryos, slide warmer, light source, embryo dissecting microscope and slide warmer behind embryos dissecting microscope. Note: this is the same area where the surgical transfers of embryos take place. The microscope to the right of the COOK incubator is used in the surgical transfer of embryos.
Figure 4.
Figure 4.
IVF plate preparation. Diagram of IVF plate. Circle W is the 250 ul washing drop for oocytes. Circle I, the 250 ul for IVF; Circles ZW1 and ZW2, 150 ul washing drops for the zygote embryos after IVF incubation. ZHD denotes the holding drop for zygote embryos for overnight incubation.
Figure 5.
Figure 5.
IVF embryos. (A) Separation of oocytes and cumulus cells approximately 4 hour after incubation of cumulus mass (white arrows) and capacitated sperm. (B) Fertilized oocytes collected after 4hour incubation. A fragmented oocyte is marked with a black arrow and a dead oocyte is marked with a white arrow. A normal oocyte is marked with the gray arrow. (C) Two cell embryos after overnight incubation. An unfertilized oocyte is marked with a gray arrow.
Figure 6.
Figure 6.
Marking of sperm cryopreservation straws. The 0.5 and 1 cm marks are for the loading of the sperm aliquots. The 4.5 cm mark is for the positioning of the mouse sperm aliquots prior to sealing.
Figure 7.
Figure 7.
Collection of sperm. (A) Exposed testicle (white arrow) by grabbing testicular fat pads. (B) Separation of cauda epididymis and vas deferens. (C) Cauda epididymis and vas deferens in CPA (D) Walking out the sperm from the vas deferens in CPA by making incisions in the cauda epididymis with 18gauge needle(9 O’clock) while grabbing other side of cauda epididymis with extra fine forceps (1 O’clock).
Figure 8.
Figure 8.
Loading of sperm cryopreservation straws. (A) Unloaded and marked straws attached to 1ml syringes lined up. (B) Sperm straws in goblets. (C and C’) Differential views of Cryo canister. (D The Cryo canister inserted in LN2 tank. (D) covered cryo canister in LN2tank while incubating loaded sperm straws in LN2 vapors for 30 min.
Figure 9.
Figure 9.
Set up of IVF media drops (A) MBCD drop (B) HTF-GSH drop (C) HTF drop and (D) HTF 4 well plate.
Figure 10.
Figure 10.
Collection of Cumulus Mass, CM (A) Dissection of oviduct from female donor, white arrow indicates swollen ampullae (B) Collecting oviducts in HTF-gsh plates (C and C’) Release CMs from ampullae by grabbing the ampullae with 18-gauge needle on the right side in the figure. This procedure is done the in mineral oil. White square box indicates enlarge area in C’ identifying ampullae red arrow-head. (D and D’) Released CMs dragged into HTF-gsh drop. The figure is showing CMS at the edge of the drop. White square box indicates enlarge area in D’ showing released CM by green arrow-head. (E) Collected CMs in peripheral area of HTF-gsh drop are shown in orange-head. (F) Collecting sperm from MBCD drop (G) Add sperm to CMs. White arrow indicate sperm. (H) Enlarged picture showing only the drop area demonstrating Sperm cloud spreading towards peripheral area of HTF-gsh drop shortly after adding sperm (White arrow).
Figure 11.
Figure 11.
Cryopreservation storage (A) Storage freezer. (B) Orange storage canisters. (C) Racks for sperm cryopreservation storage. (D) Racks for embryo cryopreservation storage.
Figure 12.
Figure 12.
Loading of embryo cryopreservation straws. (A) Unloaded and unmarked straw. (B) Marked and loaded straw. The plug is moved from the top of the straw to a position 75 mm from end of straw. Mark 1 is to allow air space between the sucrose and glycol/embryo layer. Mark 2 on the straw is the volume of sucrose added to the straw. Mark 3 on the straw if for the volume of glycol. (C) Loaded straw.
Figure 13.
Figure 13.
Freezing apparatus. (A) BioCool freezer. (B) Biocool freezing chamber (B) Racks used in BioCool freezer.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

  • Mice on the move.
    Harrison C. Harrison C. Lab Anim (NY). 2021 Sep;50(9):233-235. doi: 10.1038/s41684-021-00829-5. Lab Anim (NY). 2021. PMID: 34373647 No abstract available.

References

Literature Cited

    1. Ali J, Alharbi NH, Ali N. 2017. Chapter 1 Historical Background on Gamete and Embryo Cryopreservation. Nagy Z, Agarwal A (eds0 Cryopreservation of Mammalian Gametes and Embryos. Methods in Molecular Biology, vol 1568. Humana Press, New York, NY: 10.1007/978-1-4939-6828-21 - DOI - PubMed
    1. Austin CR 1951. Observations on the penetration of the sperm into the mammalian egg. Aust. J. Sci. Res, 4: 581–596. doi: 10.1071/bi9510581 - DOI - PubMed
    1. Austin CR 1952. The ‘capacitation’ of the mammalian sperm. Nature. 170: 326. doi: 10.1038/170326aO - DOI - PubMed
    1. Awasthi PR, French CF, Sztein J, Bedigian R, Sharp JJ, Lloyd KC 2003. Frozen sperm as an alternative to shipping live mice. Contemporary Topics in Laboratory Animal Science. 42: 8–11. - PubMed
    1. Ayabe S, Nakashima K, Yoshiki A 2019. Off- and on-effects of genome editing in mouse embryos. J Reprod Dev, 65: 1–5. doi: 10.1262/jrd.2018-128 - DOI - PMC - PubMed

Key References:

    1. Ostermeier et al., 2008. Is the original reference for the Jackson Laboratory method of mouse sperm cryopreservation. See above
    1. Renard and Babinet 1984. Is a key reference on the mouse embryo cryopreservation method. It is the basis of what a lot of labs are using to freeze down mouse embryos. See above
    1. Hogan et al., 1994. Good reference overall reference on the surgical transfer of embryos. See above
    1. Longenecker and Kulkarni, 2009. Good reference on trouble shooting of mouse surgeries. See above

Internet Resources:

Protocols and additional information
    1. www.transtechsociety.org.

    2. This is the website for the International Society for Transgenic Technologies (ISTT). It offers information related to items described in this manuscript, but also provides information on a variety of topics related to the field of transgenic technology

    1. http://card.medic.kumamoto-u.ac.jp/card/english/sigen/manual/onlinemanua....

    2. This is the website for CARD related techniques and methods.

    1. www.jax.org.

    2. The Jackson Laboratory provides workshops in the cryopreservation of mouse embryos and sperm.

    1. www.infrafrontier.eu.

    2. (INFRAFRONTIER mouse disease models) This website is related to the European Mouse Mutant Archive (EMMA) and is another useful website in mouse cryopreservation protocols and other useful resources. Genetically engineered mice can also be found here.

Finding genetically modified mice
    1. www.findmice.org.

    2. International Mouse Strain Resource (IMSR). This is an important resource for finding various mouse models that are available from various repositories IMSR

    1. www.mmrc.org.

    2. This the website for the Mutant Mouse Resource & Research Centers (MMRC). This a website supported by the NIH. It is another useful resource in finding previously genetically engineered mice.

    1. www.informatics.jax.org.

    2. Mouse Genome Informatics (MGI) Another website useful relating to genetically modified mice.

    1. https://mus.brc.riken.jp/en/

    2. RIKENBRC Experimental Mouse Division

Shipping of Biological Samples
    1. www.saftpak.com.

    2. Helpful with the shipment of biological samples using liquid nitrogen and dry ice. Company offers courses in the safe transport of infectious substances, biological samples, dry ice, liquid nitrogen, and related materials.

    1. www.aphis.usda.gov/aphis/ourfocus/animalhealth/export.

    2. Website of the Animal Plant and Health Inspection Service of the USDA. Information available on information of importation and exportation of animal related products.

LinkOut - more resources