Introduction and considerations
An undesired health status of a research animal colony may interfere with the scientific outcome from an animal study. It may also pose problems for import/export of animals between animal facilities. NTS may assist with removal of a large majority of pathogens.
In certain cases, when the colony has (or are expected to have) severely hazardous pathogen(s), NTS may not assist with rederivation as the risk of bringing these animals to our facility will be considered to large (e.g. the case for a few viruses, including parvovirus). In such cases, recovery from frozen embryos (embryo recovery) or sperm (in vitro fertilization) will represent better alternatives.
The rederivation service includes special pathogen testing of foster mothers and offspring. Animals will be delivered with a health certificate tested for the absence of any pathogens listed by FELASA.
Benefits
- Mice produced will be delivered with a health certificate confirming SOPF status.
Requirements and time frame
- A satisfactory health report that allows transfer of the animals to our facility.
- Four males 14-20 weeks of age - heterozygous or homozygous for the transgene. Proven breeders are recommended.
- Available for most strains. Cost will be higher for strains known to be difficult to superovulate, fertilize and/or implant.
- Embryos will be collected and implanted into pseudopregnant foster mothers. Offspring will be separated at 3 weeks of age, biopsies taken and provided to customer. NTS expect confirmation of genotyping within a reasonable time (1 week).
- Foster mother and offspring will be tested against the presence of pathogens according to FELASA standards. Contact NTS for a complete list of screened pathogens.
- The project is typically completed 9-12 weeks after receiving stud males.
Technical service performed by NTS
- NTS is responsible for the animal procedure permit (FOTS application).
- Planning and coordination of the project.
- Copulation training of males.
- Superovulation of females, mating overnight, dissection of oviducts and collection of embryos.
- Implantation into pseudo pregnant foster mothers, separation of offspring and collection of biopsies.
- Customer will receive biopsies in house. If personal pick-up is not an option, transport will be charged customer.
- Sample collection and coordination of pathogen screening.
Guarantee
- Under certain conditions, such as when fewer than three offspring are born, NTS will repeat the procedure once for free.
Service Fees
Estimated costs for the above service can be found on our Service Fees page.
Extended description of the rederivation procedure
Planning and considerations
If the genetically modified (transgenic) mice 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 particular unwanted pathogens.
Rederivation can be performed with hetero-, hemi- or homozygote transgenic embryos:
Alternative 1:
Collection of hemi- or heterozygote embryos. NTS will order wild-type females from a commercial vendor, superovulate these prior to mating with transgenic males, and collect embryos the next day.
Alternative 2:
Collection of homozygote embryos. Females from internal breeding is 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 enough females (>4 females) can be delivered within the same week. Two breeding cages (trio mating) are recommended to avoid any delays.
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 4 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 on the morning of day 0.5 dpc. They will then be brought to another location were the embryos will undergo several washing steps.
Embryo implantation and pathogen testing
The obtained embryos will be introduced into the oviduct of one/several pseudo pregnant foster mother depending on the number of embryos collected. Embryos will develop to term (~20 days). Offspring will be separated at three weeks of age and biopsies taken. Samples for pathogen testing will be taken from both foster mothers and offspring and sent to a commercial health monitor provider. Animals will not be delivered to the investigator before the health monitoring is completed and proven negative.
The embryo implantation procedure is occasionally unsuccessful. If three or fewer offspring are recovered, NTS will repeat the procedure without additional cost.
Service provided by NTS and 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 (if required).
- House foster mothers and vasectomized males
- Arrange plug-mating to provide pseudopregnant foster mothers.
- Implant into pseudopregnant foster mother.
- Separate foster mother and offspring.
- Take ear biopsies from offspring.
- NTS covers cage cost for donor females, foster mothers, and all procedures until offspring are 6 weeks of age.
- Donor females are terminated in the procedure. Males (studs) may be returned to the customer after completion of the project.
Costs not included
- Costs for housing of donor males (studs).
- Donor males will be charged by the animal department (internal users).
- For external customers, NTS may house animals under our FOTS permit and the cost is added to project costs.