Umeå transgenic core facility (UTCF) is housed within UCCB and is a fully integrated platform for generation, maintenance and DNA analysis of genetically modified animals. The transgenic facility performs blastocyst injections, embryo transfer, in vitro fertilization, and sperm cryopreservation.
Sleepy baby rats
ImageClient responsibilities
provide tissue samples or prepared DNA
provide primer sequences and protocols for each PCR
provide ordering form
UTCF responsibilities
prepare DNA (if client has not)
run PCR and visualize the result with agarose gel electrophoresis
send result by email, complete with gel picture within 5 working days after we receive clients samples and ordering form
The production of knock-out mice involves the microinjection of targeted embryonic stem (ES) cells into the 3.5 day old embryo, the blastocyst. The introduced ES cells will integrate within the embryo, resulting in chimeric mice where some cells in each tissue are derived from the ES cells. If the germ cells consist of the targeted ES cells, the mutation can be passed on to the progeny.
As customer at UTCF you must provide the targeted ES cells, with a health report showing that they do not carry any mouse pathogens. UTCF will then thaw a vial of each clone to be injected, expand the cells and pass them once. After that we will perform the blastocyst injection, which includes the following steps:
Collection of blastocysts
In order to be able to visualize chimeric mice by coat color, it is advisable to use a blastocyst donor strain different in coat color to that of the ES cell strain.
Similar to the procedure for pronuclear injections, 15 female mice start to superovulate after two injections of gonadotropins and then mated with stud males. The blastocysts are harvested by flushing the uterine horns from plugged females at 3.5 days post coitum.
Microinjection of ES cells into blastocysts
The harvested blastocysts will be injected with 12-15 ES cells into the blastocoel cavity. As the embryo develops, the introduced ES cells will integrate within the recipient embryos inner cell mass to form a new chimeric embryo.
Transfer of injected embryos to pseudopregnant females
In order for the injected embryos to develop to term, they are transferred into the uteri of pseudopregnant female recipients. These are females mated with appropriate timing to vasectomised (sterile) male mice. Once born, it is possible to determine the chimeric mice by coat color which will show as patches of different color in the fur. However, this is not evidence of ES cell integration within the germ cells of the mouse. Germ line transmission can only be determined by breeding the chimeras, and analysing the progeny.
Client responsibility:
Consult with UTCF coordinator
Provide 1 vial of prepared ES cells, 1 or 2 clones.
We also need the following information for each vial of cells:
Genetic background of the ES cells (what mouse strain were they derived from)
Approximate number of cells per vial.
Type of medium in which the cells were cultured before freezing.
Health report proving the ES cells free from pathogens
Provide ordering form and approved ethical permission together with ES cells
Screen resulting pups for chimeras (if not seen by coat color) or this can be done by the Genotyping facility.
UTCF responsibilities:
thaw and prepare the ES cells for injection
prepare blastocysts
inject ES cells into blastocysts
transfer injected blastocysts into a minimum of 6 foster females, or 3/clone if 2 clones were injected.
house born pups until weaning (around 4 weeks)
Mouse strains used:
Our blastocyst donors are C57Bl6 females mated with C57Bl6 males. If the client requests a different donor strain, UTCF will purchase this and any additional costs will be passed on to the client.
Client responsibilities:
consult with UTCF coordinator
for re-derivation of a mouse strain, provide at least 5 mating pairs for egg donation
UTCF responsibilities:
Dissect oviducts in order to obtain fertilized eggs
implant embryos into foster females, as many as possible from the number of eggs provided
Provide at least one born litter from the foster females
For order of Embryo transfer always consult with the UTCF coordinator.
Client responsibilities:
Consult with UTCF coordinator
Provide frozen sperm, or male(s) from which sperm will be collected
Provide females for superovulation
UTCF responsibilities:
Collect eggs from 10-20 B6/CBA females, or other background
Perform IVF using the provided sperm
Perform embryo transfer with the fertilized embryos, up to 6 embryo transfers/strain
House born pups until weaning
Is nowadays not performed by UTCF, but we can help with the contact with another facility.
Client responsibilities:
Consult with the UTCF coordinator
Provide 2 males, age 10 weeks to 6 months
UTCF responsibilities:
Freeze sperm from the males, 10 straws/male
Perform an IVF as a quality control of the frozen sperm, 1 straw/male will be used
Perform embryo transfer after IVF to ensure that live pups can be produced
Store the frozen sperm in liquid nitrogen
Blastocyst injection: 32 500 SEK
Embryo transfer: 6 500 SEK
In vitro fertilization: 6 500 SEK
Cryopreservation of sperm: 5 000 SEK; annual freezer cost 500 SEK
Genotyping: cost per hour: 440 SEK + reagents and consumables. Estimated time for 30 PCR samples =3,5-4 hours
Note, the prices apply to researchers affiliated to Umeå University.
Contact UTCF coordinator
Specific questions about genotyping