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Cryopreservation of Mouse Lines

The generation of genetically engineered mice is costly and time-consuming. Because of this investment, it would be wise to bank embryos of a valuable mouse strain to guard against the possibility of losing the line in case of an infection or disaster. In addition, at the end of a project, it is often desirable to maintain a line in the eventuality that it will be required in the future. In this instance, it can be costly to maintain the line as well as take up valuable animal space. To accommodate investigators in either of these instances, the Genetically Engineered Mouse Facility (GEMF) offers mouse cryopreservation services.

Standard Embryo Cryopreservation

We will cryopreserve 200 embryos for heterozygous males mated to wild type females at morula stage (for those strains amenable to this procedure) or 100 embryos for homozygous males. Approximately 200 or 100 two-cell embryos or compacted morula (depending on zygosity) from the mouse line of interest will be collected by the GEMF staff, cryopreserved and stored in liquid nitrogen. A minimum of 10 males that are between 8 weeks and 6 months of age are required. If the investigator wants to freeze homozygotes, females for superovulation will need to be provided. For C57BL/6 and 129 strains, females should be 21-25 days of age at use. We generally average 15 good morula from a C57BL/6 or hybrid, non-transgenic, superovulated female that is mated to a fertile male. We will apply a 10% discount for supplying proven male breeders to be demonstrated as follows: Age = three to six months; breeding records indicate two recent litters of more than six pups. If mice can be frozen as heterozygotes, the GEMF will obtain non-transgenic females of the correct age. The GEMF will superovulate the females for both homozygous and heterozygous strains. To verify the success of the procedure, one straw per freeze will be thawed, and the morula cultured overnight to the blastocyst stage. If the mouse line is unable to be cryopreserved as embryos, we will only freeze sperm from the line. There are outside sources that we can recommend for recovery of the line from the frozen sperm if this proves necessary.

Cost

$1100/line/session for MD Anderson investigators; $3000/line/session for external investigators

Time Line for Standard Embryo Cryopreservation

Day 1

  • Administer PMS to females
  • Administer HCG to females and mate
  • Transfer females to a separate cage

Day 6

  • Flush oviducts to collect morula and freeze
  • Thaw one straw for overnight culture

Day 7

  • Count blastocysts in overnight culture

To Obtain Service from the GEMF

Sperm Cryopreservation

We now utilize the EMMA protocol initially published by Naomi Nakagata as our method of sperm cryopreservation [Ostermeier GC et al. 2008. PLoS One 3(7):e2792]. The investigator will need to supply two male mice that have been previously mated according to the schedule set by the GEMF upon submission of the service request form. Eleven straws of sperm per male will be frozen and stored in the GEMF. To test the integrity of the procedure, we will use one straw from each male to perform IVF with wild type oocytes, which will be incubated to the two-cell stage to establish fertilization rates.

Cost

$730/line for MD Anderson investigators; $2000/line for external investigators

Time Line for Sperm Cryopreservation

Day 1

  • Two to four males are set up in mating to non-essential females supplied from the PI's colony

Day 5

  • Males are unmated to allow for sperm recovery

Days 10 & 11

  • Males are brought to the GEMF Cryo lab for sacrifice and sperm recovery and crypreservation

Approx. Day 17

  • One sperm sample per male is thawed and mixed with unfertilized oocytes to allow for in vitro fertilization. After four hours, oocytes are washed and allowed to incubate overnight

Approx. Day 18

  • Two-cell embryos resulting from the IVF are counted and the fertilization rate for the sperm sample is calculated. Project is complete

To Obtain Service from the GEMF

  • Complete and submit a service request form for sperm cryopreservation. (This form can be faxed to Jan Parker-Thornburg at 713-792-6028.)
    Service request form for MD Anderson investigators (pdf)
    Service request form for external investigators (pdf)
  • A GEMF technician will contact you with details on the mating protocol prior to the cryopreservation procedure.
  • You will bring the properly mated males to the Cryo lab at a time pre-arranged with the GEMF technician.
  • You will be notified when the project is complete.

IVF-Cryopreservation

As it can often be difficult for investigators to produce 15-20 fertile males for standard embryo cryopreservation, we also offer the option of an IVF-Cryopreservation procedure. For this procedure, we will require two to four optimally fertile males. The males will be sacrificed to obtain sperm that is then mixed with oocytes in vitro. Two-cell embryos resulting will be vitrified and stored for future use. While this procedure is much easier on the investigator, there will often be long waiting times for this service due to the amount of technical time required. As in the standard embryo cryopreservation procedure, we will archive 200 embryos from a heterozygous x wild-type mating and 100 embryos from a homozygous x wild-type mating. All procedures will be tested by thawing a small aliquot of embryos and determining survival.

Cost

$2000/session for MD Anderson investigators; $5450/session for external investigators

Time Line for IVF-Cryopreservation

Day 1

  • Two to four males are set up in mating to non-essential females supplied from the PI's colony

Day 5

  • Males are unmated to allow for sperm recovery

Days 10 & 11

  • Males are brought to the GEMF Cryo lab for sacrifice and sperm recovery, IVF and cryopreservation

Approx. Day 17

  • One sperm sample is mixed with unfertilized oocytes to allow for in vitro fertilization. After four hours, oocytes are washed and allowed to incubate overnight

Approx. Day 18

  • Two-cell embryos resulting from the IVF are counted and the fertilization rate for the sperm sample is calculated. All two-cell embryos are vitrified and maintained in liquid nitrogen. One small group of embryos is thawed to determine survival rates. After successful vitrification of 200 embryos (100 embryos if a homozygous male is used), project is complete. (Note: If the requisite number of embryos is not obtained, the project will be repeated at an additional session-cost to the investigator.)

To Obtain Service from the GEMF

  • Complete and submit a service request form for IVF-cryopreservation. (This form can be faxed to Jan Parker-Thornburg at 713-792-6028.)
    Service request form for MD Anderson investigators (pdf)
    Service request form for external investigators (pdf)
  • A GEMF technician will contact you with details on the waiting time until the project is started, and the mating protocol prior to the cryopreservation procedure.
  • You will bring the properly mated males to the Cryo lab at a time pre-arranged with the GEMF technician.
  • You will be notified when the project is complete.

© 2014 The University of Texas MD Anderson Cancer Center