Genetically modified mouse models have become a key tool in basic and biomedical research. The ability to engineer the mouse genome has greatly transformed biomedical research in the last decade. Crucial for this technology is the ability to control the expression of genes of interest. It is possible to increase or decrease gene expression, or eliminate the expression of a gene completely. Transgenic and gene knockout/knockin technologies have become important experimental tools for assigning functions to genes at the level of whole complexity of organism, creating models of genetic disorders, evaluating effects drugs and toxins, thus helping to answer fundamental issues in basic and applied research.
Our service include pronuclear microinjection of DNA constructs into mouse zygotes for the production of transgenic founders; microinjection of targeted ES cell lines into morulas (8-stage cell embryo) to produce chimeric mice; mouse archiving (cryopreservation of embryos and sperm); and recovery of live mice from cryopreserved embryos and sperm, analysis of sperm viability, rederivation of mouse strains and lines, and others. We also provide consultation and assistance services, and information on the design and use of genetically modified transgenic mice. All of our services are also available to external institutions and researches irrespective whether they are from academic or profit organizations.
- ContactOpen or Close
For more details about our services, consultations, price quotations or request submissions please contact us at following email addresse: email@example.com Your message will be guided to a respective person in charge.
Radislav Sedlacek, PhD (Head of the Module)
Petr Kasparek, (Deputy Head of Module, Head of Targeting Unit)
Jana Kopkanova, MEng. (Head of Genotyping and Breeding Unit, Cryopreservation & EMMA/Infrafrontier Services)
Irena Jenickova, PhD (Head of ESCs Technologies and Production Unit) – maternity leave
Transgenic and Archiving Module of the Czech Centre for Phenogenomics
IMG / BIOCEV
252 50 Vestec
Tel: (+420) 325 873 222 (Petr Kasparek)
Tel: (+420) 325 873 256 (Jana Kopkanova)
- Mouse Models generation & genetic engineering servicesOpen or Close
Pronuclear and cytoplasmic injections.
Generation of transgenic mice by pronuclear injection (PNI) of fertilized oocytes is the one of the most common and convenient way to make a transgenic mouse although it has also some shortcomings such as multiple transgene copies, occasional mosaic founders, expression levels do not correlate well with copy number, etc. Despite of these limitations PNI is often the quickest possibility to realize your project.
We offer also generation of transgenic animals using BACs (bacterial artificial chromosomes) and transposons. BAC transgenes usually have several advantages over transgenes produced by classic constructs; they give more physiological expression levels, are not susceptible to epigenetic inactivation, and give expression in proportion to copy number.
Knock-out/knock-in mouse generation.
TgU can culture and/or microinject ES cells for customers who have either generated targeted clones in their lab or obtained ES cell clones from another source. The provided ES cells are microinjected into the eight-stage cell embryo using laser-assisted technology.
TgU offers also generation of genetically-modified mouse mutants using TALE- and Zinc-finger (ZFN) nucleases and CRISPR/Cas9 nucleases. Using those technologies, we are providing design, validation and a mouse model generation on C57Bl/6NCrl background.
- Rederivation / ReanimationOpen or Close
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.
- Archiving ServicesOpen or Close
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 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.
- Generation of Mouse MutantsOpen or Close
Transgenic mice (pronuclear injection)
Generation of transgenic mice will be performed by pronuclear injection (PNI) of fertilized oocytes. This technique is the most common and convenient way to make transgenic mice although it has also some shortcomings such as multiple transgene copies, occasional mosaic founders, expression levels do not correlate well with copy number, etc. Despite of these limitations PNI is often the quickest possibility to realize your project.
In addition to the PNI of classic construct we offer also generation of transgenic animals using BACs (bacterial artificial chromosomes) and transposons. BAC transgenes usually have several advantages over transgenes produced by classic constructs; they give more physiological expression levels, are not susceptible to epigenetic inactivation, and give expression in proportion to copy number.
To generate transgenic mice we use donor oocytes derived from superovulated C57BL/6N mice. Investigator/customer has to provide DNA-construct prepared according to the protocol (see the Protocols section). Purity of the DNA is a critical point in the generation of transgenic mice.
Generation of knockout or knockin mice – ES cell injection
The TgU can culture and microinject ES cells for customers who have either generated targeted clones in their lab or obtained ES cell clones from another source. The provided ES cells are microinjected into the eight-stage cell embryo using laser-assisted technology. The injected embryos are then surgically implanted into the oviducts of recipient females (foster mothers). We use C57BL/6 mouse colony for microinjection of 129-derived ES cells. Customers with ES cell lines derived from other strains should contact the head or supervisor of TgU for additional information.
As the maintenance of ES cells in a non-differentiated state is largely dependent on how the cells are treated/handled in the cell culture, we cannot guarantee that microinjections will result in the generation of chimeric mice or that chimeric animals that are produced will be germ-line competent when working with ES cell clones cultured outside our facility.
- Request forms – Transgenic and Archiving Module servicesOpen or Close
For the time being only request forms for download are available. Please use email firstname.lastname@example.org for submitting your requests.
TALEN CRISPR generation request form TAM-CCP PDF DOC
Pronuclear or Cytoplasmic injection request form TAM-CCP PDF DOC
Sperm freezing request form TAM-CCP PDF DOC
IVF rederivation request form TAM-CCP PDF DOC
Embryo freezing request form TAM-CCP PDF DOC
ES cell injection request form TAM-CCP PDF DOC
Embryo transfer and rederivation request form TAM-CCP PDF DOC
Breeding of mouse strains request form TAM-CCP PDF DOC