Genotyping and Breeding

Genotyping and Breeding, Cryopreservation & EMMA/Infrafrontier Services

We represent a Czech node of EMMA (European Mouse Mutant Archive), a non-profit repository for the collection, archiving (via cryopreservation) and distribution of relevant mutant mouse strains essential for basic biomedical research. More info

Our services comprise of:

  • embryo and sperm cryopreservation, and reanimation of strains from frozen material;
  • ovary transplantation;
  • rederivation/ cleaning of mouse/rat strains;
  • genotyping service, and
  • import/ export arrangements (in collaboration with the animal facility module).

Standard Services Archiving Services

Sperm Cryopreservation

Cryopreservation of mouse sperm (sperm freezing) is used to archive male germ cells. Each cryopreservation procedure includes harvesting, visual assessment, washing, and cryopreservation of sperm using thin-walled straws. We are routinely freezing 11 straws; the 11th one is a control straw that is used to check the viability of sperms. Aliquots in straws, marked by ID number, are stored in goblets in bulk liquid nitrogen tanks. For best results males should be 8 to 35 weeks old and proven breeders that have been housed apart from females for at least three days. Requesters should ensure that they have at least two proven male breeders remaining in their colony to continue live breeding until the viability of frozen sperm is confirmed. Sperm cryopreservation should be used when you want to preserve a mouse line with male germ cells and can recover the line using wild-type donor oocytes of the same strain/line.

Sperm cryopreservation is cost effective compared to embryo cryopreservation. Recent methodological progress on archiving methods have resulted in improved and reliable fertility rates of cryopreserved mouse sperm following thaw. The quality control, i.e. generation of two-cell embryos or live born mice including animal housing, will be charged to the requester additionally.

Embryo Cryopreservation

Embryo archiving provides long-term storage of mouse lines and strains. Cryopreserved embryos (and sperm) can also be sent to other institutions and collaborating scientist without the complicated regulations requested for shipment of live animals.

While this is a robust, reliable cryopreservation method, it is relative costly. Not all mouse strains are suitable for embryo cryopreservation: embryos from some strains do not withstand the freeze/thaw process. However, embryo cryopreservation in C57BL/6 and B6 hybrid strains works very well.

Retrieval of mouse strains/lines is reliable and the cost of reanimation is cost effective in comparison to the long-term breeding.

TgU established the embryo freezing in 2010 to store or ship your lines. Please contact us if you need this service, or expect to need it in the future.

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Custom services Rederivation / Reanimation

Mouse strains and lines are often infected with various pathogens in conventional holding and these infections may potentially interfere with your experiments and the outcome of the experiment could be changed or biased. Rederivation (cleaning) of an infected mouse strain will be typically performed by super-ovulation of females, mating with males, and isolation of fertilized embryos (E0.5 to E2.5). Embryos will be washed and transferred into oviduct of pseudopregnant SPF foster mice. If necessary, for some mouse lines and strains natural mating will be carried out to obtain suitable embryos for the transfer. Mice of CD-1(ICR) strain are used as pseudopregnant foster mothers.

Internal investigators (IMG and IBT members) normally perform super-ovulation, mating, and plug-check on their own. Females with plugs will be delivered to TgU for embryo isolation.

External investigators (non-IMG and non-IBT members) should provide 10 proven breeder males, 8-9 weeks of age and 10 females, 4-8 weeks of age. The import and acceptance of these mice have to be arranged with the head of IMG Animal Facility. In Vitro Fertilization (IVF) could be also used to rederive infected strains/lines.

Rederivation and Import of Mouse Lines Using Frozen Embryos

Rederivation of a mouse strains/lines can be also performed by transfer of embryos from a frozen stock. Such embryos will be transferred into the oviduct of foster mothers. Investigators/customers must inform TgU about the actual date of embryo delivery, the mouse strain/line, and about the freezing-thawing procedure in advance.

In Vitro Fertilization (IVF) and Reconstitution of Cryopreserved Sperm

In vitro fertilization is used to retrieve mice strains/lines from cryopreserved sperm, when there is a behavioral or physical obstacle to mating, or when fertilization needs to be observed. Donor oocytes are usually taken from C57Bl/6 females. TgU IVF service includes IVF from both fresh and frozen sperm. Using the frozen sperm, the sperm samples will be thawed, IVFs performed, and fertilized eggs transfered into suitable SPF foster mothers.

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