Embryo cryopreservation

Summary

Cryopreservation of a genetically modified (transgenic) mice line provides an archive where genetic drift no longer occurs and safeguards the line against elimination due to infection, disease, breeding failure or other unforeseen incidents. Cryopreservation also serves as an ethical as well as an economical choice for transgenic lines maintained solely to prevent loss of the transgenic line by freeing up animal facility space and reduce breeding costs, respectively.

NTS offers cryopreservation of both sperm and embryos. For sperm cryopreservation, please go here.

Benefits

  • Protection against genetic drift or spontaneous loss of phenotype.
  • Saves resources when colonies are not in use.
  • Allows easy distribution to collaborators or other institutions.
  • Allows for a quick rederivation into an SOPF barrier.

Requirements and time frame

  • Satisfactory health report for transfer of the animals to our facility.
  • 8 males 14-20 weeks of age - heterozygous or homozygous for the transgene.
  • Proven breeders are required.
  • Embryos will be collected and grown to 2-cell stage prior to cryopreservation.
  • Embryos will be collected for >3 weeks, depending on fertilization efficiency.
  • Project is typically completed 6 weeks after receiving stud males.
  • Available for most strains. Cost will be higher for strains known to be difficult to superovulate, fertilize and/or implant.

Technical service performed by NTS

  • NTS is responsible for the animal procedure permit (FOTS application).
  • Planning and coordination of the project.
  • Superovulation of females, mating overnight, dissection of oviducts and collection of embryos.
  • A minimum of 200 2-cell (8 tubes) heterozygous embryos will be frozen when supplied with homozygous males.
  • A minimum of 250 2-cell (10 tubes) heterozygous/wild type embryos will be frozen when supplied with heterozygous males.
  • Frozen tubes will be distributed into two separate liquid nitrogen tanks.

Quality control (QC)

  • Implantation of embryos from one of the frozen batches into pseudopregnant foster mothers.
  • Foster mother may be culled in the days preceding birth and biopsies taken - to avoid any chance of cannibalism.
  • Customer will receive biopsies. If in personal pick-up is not an option, transport will be charged customer.
  • NTS expects confirmation of genotyping within a reasonable time (1 week).

Costs cover the following

  • Purchase and transport of donor females from vendors.
  • Up to four rounds of freezing. Extra rounds will add >4000 NOK pr. round (NTS will charge for the purchase of additional donor females).
  • Quality control (QC) – with birth of >1positive offspring from a test thaw.
  • Cage cost for housing of donor females, foster mothers and offspring until 6 weeks of age (if customer want to confirm viable animals).
  • Embryos cryopreserved for first year in 2 separate tanks included in price.
  • Additional years of storage available for 500kr/strain/year.

Service Fees

Estimated costs for the above service can be found on our Service Fees page.

Extended description of the procedure

Planning and considerations

If the genetically modified (transgenic) mouse line is housed outside our animal facility (currently KPM at UIO), an updated health report is needed to determine if the mice can be temporarily transferred to and housed into our animal facility.

The standard arrangement uses embryo donors from a commercial supplier. Please contact NTS to discuss details if this appears not to be an option for you. Investigators may alternatively choose to breed their own animals and superovulate locally to generate the necessary number of embryos. This should be done in close collaboration with the NTS staff to assure success. This choice can e.g. be practical if the transgenic line is housed in an animal facility with a history of certain unwanted pathogens.

Embryo cryopreservation can be performed with either hetero-, hemi- or homozygote transgenic embryos:

Alternative 1:

Collection of hemi- or heterozygote embryos. NTS will order wild-type females from commercial vendors, and superovulate these prior to mating with transgenic males for embryo collection the next day.

Alternative 2:

Collection of homozygote embryos. Females from internal breeding are used for superovulation and mating with transgenic males. As long as the breeding performance is not affected by the genetic modification, mice of any genotype might be used with this arrangement. However, it is essential that a high number of females (>8 females) can be repeatedly delivered each week, over a 3-4 weeks period, to avoid any delay of the project (NTS or the customer will set up two-three breeding cages (trio) every week for three weeks, in total >6 breeding cages).

Provided by the customer

The breeding scheme required for generation of the embryos depends on genetic background and will be evaluated for each individual case. At a minimum, the researcher needs to provide multiple proven breeder males to obtain fertilized embryos. NTS recommends having 8-10 proven fertile males containing the genetically modified allele (studs). If female donors cannot be obtained from a commercial supplier, the birth and delivery of donor females must be timed well in advance with the NTS staff.

Embryo fertilization and harvesting

The procedure is roughly as follows. Young female mice (donors) are treated with hormones to induce superovulation prior to mating. Following two rounds of hormone administration (PMS followed by HCG), females are mated over night with males containing the genetically modified allele. The reproductive tracts from the mice are aseptically isolated in the morning of day 0.5 dpc. They will then be brought to another location were the embryos will be recovered and cultured overnight to ensure that they are able to divide to the two-cell stage before frozen down. Alternative mating strategies to the above can be arranged.

Embryo cryopreservation

We use a method called equilibrium cryopreservation, or slow freezing as compared to vitrification. In short, the embryos are cooled down to low subzero temperatures (-40°C) in the presence of moderate concentrations of a cryoprotectant. During this process embryo dehydration occurs. The embryos will be stored in liquid nitrogen. Optimally ≥200 embryos divided on >8 tubes/straws will be frozen down to ensure a reliable later reestablishment of a breeding colony.

The embryo harvesting and cryopreservation steps will be repeated until the desired number of embryos are frozen down (usually ~three successive rounds/weeks).

Of note: All preimplantation stages can be frozen down and revived after prolonged storage. The method chosen for revival will depend on the circumstances for cryopreservation of the embryos. Whenever possible we would recommend preservation at the two cell stage. It is reasonably simple to get a good yield and later stages can be produced through in vitro maturation.

Quality check

The ability to reconstitute a stock from frozen embryos is tested at the time the strain is frozen down (for description see the embryo recovery procedure). One frozen tube is thawed to ascertain that they can develop to term. Biopsies are given to the customers for verification of the presence of the genetically modified allele.

Embryo recovery

A sufficient number of embryos will be thawed (usually one frozen tube). The obtained embryos will be transferred to pseudopregnant foster mothers. Blastocysts will be implanted in the uterus of pseudopregnant females upon embryo thawing. All other stages will be introduced into the oviduct. Biopsies will be taken from all resulting offspring before they are transported to the investigator (earliest at 3 weeks of age). The embryo recovery procedure is occasionally unsuccessful. If three or fewer living animals are recovered, NTS will repeat the procedure without additional cost.

Storage fees

A yearly storage fee is charged for all frozen strains stored in the NTS repository. NTS may also store frozen embryos provided by customers. For each stocked genetically modified line, tubes containing embryos are split on two separate liquid nitrogen tanks, as a safety precaution. NTS inspects these tanks and refill with liquid nitrogen at least once a week.

Note: Due to the strict routines for preparation and conservation of cryopreserved embryos on liquid nitrogen, NTS can only guaranty successful recovery of embryos that are both frozen down and stored by us.

Limited guarantee

Investigators can request recovery of strains at any time. In most cases, the process can be successfully repeated several times from one stock of cryopreserved embryos. However, NTS guaranties only one successful recovery from a cryopreserved strain. Whenever the number of frozen embryos stored at NTS drops below 200 (e.g. due to thawing of embryos), NTS will still keep the remaining embryos, but will no longer guaranty that the line can be recovered from this insufficient stock of frozen embryos.

If the customer wants to cryopreserve embryos to distribute the strain, it is advised to freeze down additional embryos for that purpose.

Service provided by NTS included in the cost estimate

  • Approval for the procedure (FOTS application).
  • Organize transport of animals.
  • Arrange for single housing of genetically modified males (studs).
  • Train studs (arrange mating with female prior to the procedure).
  • Ordering of donor females (cost is included in the cost estimate).
  • Purchase of required hormones.
  • Superovulation and set up mating to obtain embryos.
  • Dissect out oviducts and isolate embryos.
  • Culture overnight and cryopreserve embryos.
  • House and provide pseudopregnant foster mothers.
  • Test thaw and implant into pseudopregnant foster mother.
  • Separate foster mother and offspring.
  • Take ear biopsies from offspring.
  • NTS covers cage cost for purchased donor females, foster mothers, and all steps until offspring are 3 weeks of age (if customer wants to confirm viable animals).
  • Donor females are terminated in the procedure, but males may be returned to the customer after completion of the project

Costs not included

  • Costs for housing of donor males.
  • Donor males will be charged directly from animal department.
  • For external customers, NTS may house animals under our FOTS permit and the cost is added to project costs.
Published Nov. 28, 2017 2:29 PM - Last modified Mar. 27, 2023 10:41 AM