The Centre for Phenogenomics
The Centre for Phenogenomics

Cryopreservation & Recovery Services

With monitored storage facilities at two separate locations to keep your valuable mouse models secure and the extensive experience to recover them successfully, we can help you manage your strains.

The Canadian Mouse Mutant Repository (CMMR) is embedded in The Centre for Phenogenomics to provide cryopreservation, recovery, and distribution of mouse lines. More information about the CMMR and its publicly available resources can be found at www.cmmr.ca.

Cryopreservation

Cryopreservation archives mouse lines for long-term storage and protects against genetic drift and loss.

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Important Notes:

  1. Frozen samples are divided and stored at two different sites in liquid nitrogen storage vessels under 24-hr surveillance to ensure safety of the frozen archive.
  2. The Requester should ensure that they have sufficient proven breeders remaining in their colony to maintain the line until quality control of cryopreserved samples is complete.
  3. Oocyte, embryo and sperm harvest are terminal procedures; however, collection of other tissues for experimental purposes can be arranged.
  • Sperm Cryopreservation preserves only the male germline and is suitable for mouse lines on commercially available inbred or outbred backgrounds.

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    • Each session of Sperm Cryo includes harvest from a single male, pre-freeze assessment for viability, concentration, and motility, washing, and cryopreservation. Straws (~10-12/male) are stored in liquid nitrogen storage tanks at two separate locations to ensure safety of the archive.
    • For best results, 11 to 20-week old males that have been singly housed for three to five days should be used.
    • For each mouse line cryopreserved as sperm, a post-thaw assessment of sperm viability and motility from at least one male is done as the minimum quality control.
    • Additional quality control may include in vitro fertilization (IVF) with strain-matched oocytes to determine fertilization rates and/or transfer of the derived embryos to pseudopregnant recipients to assess the number of live born mice. If IVF is performed, any embryos not used for transfer are cryopreserved and stored as additional stock for the mouse line.

  • Basic Embryo Cryopreservation is used when both the male and female germlines must be preserved, such as for recombinant inbred lines, male sterile or lethal lines, strains with unique genetic backgrounds, or when homozygotes need to be cryopreserved.

    Read More about basic embryo cryopreservation

    • Timed mating between stud males and superovulated females are set up to provide up to 10 mated 2.5-dpc pregnant females for embryo harvest and cryopreservation. Superovulation protocols are age-dependent and for most strains effective in young females (3-4 weeks of age). Read More
    • Requesters perform superovulation and mating in their own colonies and deliver plugged females to TCP. Alternatively superovulation, mating, and plug checking for cryopreservation can be done by TCP staff when animals are located at or imported into TCP.
    • We recommend that at least 8-10 proven breeder males be available as studs for each Basic Cryo request. Depending on the number of males available, their fertility, and strain response to superovulation, up to four sessions of Basic Cryo may be necessary to produce and adequate number of embryos. Suggestions, breeding protocols and gonadotropin hormones for superovulation to assist the Requester can be provided.
    • Each session of Basic Cryo includes embryo harvest and cryopreservation from up to 10 plugged females. The number of embryos produced depends on the fertility and fecundity of the strain and whether superovulation protocols exist and/or are used before mating. Embryos are stored in straws (~20/straw) in liquid nitrogen.

  • Speed Embryo Cryopreservation is used when embryos are the desired cryopreserved archive, such as when the line will be frequently accessed or distributed, or when adequate sperm cryopreservation protocols are not available for the genetic background.

    Read More about speed embryo cryopreservation

    • In vitro fertilization (IVF) is used to produce large numbers of embryos for cryopreservation. Requesters provide two males, 8-20 weeks old, for each IVF session as sperm donors for fertilization of wild-type oocytes. Males should be singly housed for a three to five days before scheduled sperm harvest. Read More
    • Oocyte donors are purchased from commercial vendors. These wild-type strains may be used: C57BL/6J, C57BL/6NCrl, C57BL6/NTac, C3H, 129, FVB CD-1, or ICR.
    • After IVF, embryos are cultured in vitro for 24 hours then two-cell stage embryos are frozen in straws (~20/straw). The actual number of embryos produced depends on the fertility of the males provided and the strain of female oocyte donors. Depending on the genotype of the male(s), either heterozygous or a mix of heterozygous and wild-type embryos will be preserved.
    • Fees include one IVF session, 2-cell embryo harvest & cryopreservation using donor male sperm and wild-type oocytes. Quality control is extra.
    • For each mouse line cryopreserved by Speed Cryo we perform at least one session of Quality Control to Live Born. Embryos are thawed and transferred to a pseudopregnant recipient for gestation and birth. This ensures the mouse line is recoverable before it is discontinued or distributed
    • Sperm can be cryopreserved at the same time as Speed Cryo for an additional fee.

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Cryorecovery

Cryorecovery is used to re-establish a line from cryopreserved stock, either from the CMMR’s archive or from frozen stock from another repository. Transporting cryopreserved material is often less costly than live animal shipment and relieves the animal welfare concerns associated with shipping live animals.

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All cryorecovery protocols derive mice into SPF status at TCP. Requesters must have the derived mouse line added to an approved animal use protocol (AUP) in order for the derived mice to be transferred to their colony. All mouse lines stored as cryopreserved stock at CMMR have a mouse line passport in TCP LIMS. This passport can be used to add the mouse line to the relevant AUPs at TCP or can be printed as a PDF and submitted to the local Animal Care Committee (ACC) for AUP approval. Mouse lines being recovered at TCP must have an approved mouse line passport in TCP LIMS prior to recovery to conform to Animal Use Protocol requirements.

  • Sperm recovery recovers mice from frozen sperm stocks.

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    • One straw of frozen sperm is thawed and used for in vitro fertilization (IVF) with oocytes harvested from 10-20 commercially available superovulated females to generate embryos.
    • Up to 80 of the derived embryos are transferred into 4 pseudopregnant females. Additional fees apply for transfer of additional embryos. The remaining embryos are cryopreserved and stored for the investigator.
    • Tissue samples are obtained after birth for genotyping by the Requester or at TCP (additional fees for genotyping apply).
    • Recipient mice are subjected to health testing to ensure SPF status has been maintained.
    • Recovered mice are transferred to the investigator once SPF status has been confirmed.

  • Embryo recovery recovers mice from frozen embryos.

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    • Embryos are thawed and transferred into pseudopregnant recipients for gestation and delivery.
    • Fees also include communication with supplying repository, receipt & temporary storage of embryos, if applicable.
    • Tissue samples are obtained after birth for genotyping by the Requester or by TCP (additional fees for genotyping apply).
    • Recipient mice are subjected to health testing to ensure SPF status has been maintained.
    • Recovered mice are transferred to the investigator once SPF status has been confirmed.

  • Embryo and sperm import enables frozen stock to be stored at CMMR on behalf of the requester until recovery or for long-term storage.

    Read More about embryo and sperm import

    • Frozen stock should be shipped directly to CMMR so that it can be transferred to appropriate storage without delay.
    • All shipping is at the Requester’s expense and includes the return of any dry shippers to the originating facility.
    • If frozen stock is being shipped across international borders, the Requester must ensure that all paperwork applicable to animal, embryo and/or sperm import/export is in place to ensure no customs delays.

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Strain Services

Strain Services use in vitro fertilization (IVF) to rapidly expand mouse lines or rescue mouse lines that have stopped breeding by natural mating. These services require at least one fertile male carrying the desired mutation(s).

Read More about important notes

Important Notes:

  1. Fees include superovulation of commercial oocyte donors, sperm & oocyte harvest, IVF, embryo washing and assessment, transfer of ~80 embryos to 4 pseudopregnant recipients, tail biopsies, and per diems to weaning for each session.
  2. In general, each recipient will receive approximately 20 embryos and will be housed for six weeks until litters are weaned. Additional embryos may be transferred, with each 20 additional embryos requiring one pseudopregnant recipient with housing; or embryos may be cryopreserved.
  • Speed expansion generates a large number of embryos by IVF for transfer into pseudopregnant recipients to rapidly expand a colony.

    Read More about speed expansion

    • Requesters provide one or more genotype-confirmed male mice as sperm donors.
    • Superovulated commercially available females are used as oocyte donors. In some cases, females from the Requesters colony can be used to expand the colony.
    • Depending on the mouse line background and fertility of the male, up to 200 embryos can be produced from a single IVF session.
    • Unless otherwise specified at time of request, all resultant viable embryos are transferred into pseudopregnant recipients (~20 embryos/female) and listed fees include 10 embryo transfer surgeries and pseudopregnant recipients. If fewer recipients are used, charges will be reduced accordingly.
    • The requester may specify a maximum number of embryos to be transferred when the request is submitted (estimate ~40% of embryos transferred will produce live born pups). If applicable, embryos not transferred are cryopreserved for an additional charge.
    • Recipient mice are subjected to health testing to ensure SPF status has been maintained.
    • Recovered mice are transferred to the investigator once SPF status has been confirmed.

  • Mouse line rescue via IVF is used to attempt recovery of a mouse line that has stopped natural breeding.

    Read More about mouse line rescue

    • Requesters provide one or two genotype-confirmed male mice as sperm donors.
    • Superovulated commercially available females are used as oocyte donors.
    • Up to 80 of the derived embryos are transferred into 4 pseudopregnant females. Additional fees apply for transfer of additional embryos. The remaining embryos are cryopreserved and stored for the investigator.
    • On request and for an additional charge, sperm may be cryopreserved at the time of IVF so that if IVF fails, other options (e.g. intracytoplasmic sperm injection) can be explored.
    • Recipient mice are subjected to health testing to ensure SPF status has been maintained.
    • Recovered mice are transferred to the investigator once SPF status has been confirmed.

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Distribution

Distribution services allow mouse lines to be shared as frozen stock with collaborators and/or other service providers. Shipping cryopreserved stock, especially across international borders, is often simpler than live animal shipment and avoids the animal welfare concerns associated with shipping live animals.

  • Specialized dry shippers for transport of frozen embryos or sperm at liquid nitrogen temperatures ensure security during transport.
  • Communication with the Requester’s courier and the recipient facility regarding shipping logistics, export documents, and protocols ensures successful recovery of cryopreserved stock.
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Embryonic Stem Cells

Embryonic stem cells are available from the CMMR including:

  • wild-type C57BL/6NTac-derived C2 ES cells (C57BL/6NTac)

    Read More about wild-type C57BL/6NTac-derived C2 ES cells

    • C2 cells were used as the parental line for the NorCOMM targeting project.
    • C2 cells are available for use in both individual investigators’ laboratories or by Core facilities.
    • Culture and germline transmission rates for C2 ES cells were described in Gertsenstein et al., 2010.

  • NorCOMM gene trap ES cells (R1 parental line, 129-hybrid)

    Read More about NorCOMM gene trap ES cells

    • The CMMR is the repository and distribution centre for the NorCOMM gene trap ES cell library .
    • The majority of targeted genes are null alleles, however a few were targeted as conditional alleles. Many targeted lines have been converted to mouse lines also available from the CMMR.
    • Prior to distribution, targeting is confirmed by long-range PCR with primers beyond the end of the 5′ homology arm and within the targeting cassette.
    • Requests normally take 8-12 weeks for expansion and validation before lines are shipped.

  • NorCOMM targeted ES cells (C2 parental line, C57BL/6NTac)

    Read More about NorCOMM targeted ES cells

    • The CMMR is the repository and distribution centre for the NorCOMM targeted ES cell library .
    • The majority of targeted genes are null alleles, however a few were targeted as conditional alleles. Many targeted lines have been converted to mouse lines also available from the CMMR.
    • Prior to distribution, targeting is confirmed by long-range PCR with primers beyond the end of the 5′ homology arm and within the targeting cassette.
    • Requests normally take 8-12 weeks for expansion and validation before lines are shipped.

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