SummaryThe transgenic service facility is a CRM core facility that provides services on a recharge basis for making, importing, and preserving genetically-modified mice.
The facility also provides services such as teratoma assays, ES cell transfections for targeting, ES cell derivation from blastocysts and other bespoke transgenic services. Our highly experienced transgenic technicians have created a large number of new transgenic lines that have been published widely. We also provide services to other University of Edinburgh based researchers on a recharge basis.
The facility is located at the SCRM Building at Little France.
Embryo and sperm cryopreservation provides security against loss of valuable strains and a low-cost means of holding strains over long periods for future use or distribution. We offer:
- Collection, freezing and storage of sperm or embryos in liquid nitrogen. Testing of recovery by thaw and mobility check of a sample for quality control.
- Collection, freezing and storage of embryos at morula in liquid nitrogen. Testing of recovery by thaw, incubation and transfer to recipients.
ES cell gene targeting and transgenesis
The Transgenic Service offers services to aid gene targeting in ES cells as well as microinjection of targeted lines for germline transmission. We offer:
- Electroporation of DNA constructs into validated germline-competent ES cells as well as selection, expansion and freezing of targeted clones. Chromosomal DNA is returned for analysis of targeting events. We subsequently expand positive cell lines, prepare DNA for confirmation to prepare clones for microinjection. Currently the service does not offer construction of targeting vectors or Southern blot screening of clones. However, our experience team can provide support and advice for this.
- Microinjection of targeted clones. Chimeric mouse production and testing for germline via coat colour transmission. This service is available to other academic facilities and companies on a recharge basis. Please be aware that ES cells targeted in other facilities will require pathogen testing prior to injection for an additional charge.
ES cell line derivation
This service offers ES cell line derivation directly from blastocysts from wild type and mutant mice strains. This technique is highly efficient and utilising serum free media containing small molecule inhibitors (2i media).
- Immunosurgery and explant culture in 2i serum free media
- Expansion, passage and freezing of new ES cell lines in 2i media
- Conversion of 2i derived ES cells into standard serum containing media
- DNA preparation for screening of derived lines
- Mycoplasma testing of derived lines
- Distribution of early passage, germline-tested ES cells
We can derive ES cells from a wide variety of mouse strains including 129Sv and C57Bl/6. Please contact the Transgenic Service to discuss your exact needs.
Morula and tetraploid aggregation
Chimeras can be created with high ES cell contribution as a means to analyse the in- vivo developmental potential of mutant ES cell lines and primary cells.
We create these by an efficient aggregation procedure with diploid morulae. By taking an embryo at the two-cell stage and fusing the two cells by applying an electrical current, we can also produce tetraploid cells.
These tetraploid cells can form the extra-embryonic tissue (placenta etc...), however a foetus will rarely develop. When using tetraploid embryos with ES cells in a tetraploid complementation assay, the foetus is exclusively derived from ES cells.
- Collection of embryos
- Morula or tetraploid aggregation of cells with embryos
- Embryo transfer to recipient mice
- Dissection of midgestation chimeras if required
- Histological analysis if required
Rodent strain rederivation
The transfer of rodents such as mice from one facility to another often requires the rederivation of the strain through transfer of embryos via oviducts or uteri to rederive new lines as a specific pathogen-free colony.
Collection of preimplantation stage embryos from imported strains
Transfer to the uterus of appropriate recipient females and monitoring of births
Teratoma assays and tissue engraftment
To test the developmental potential of ES cells or iPS cells it is possible to surgically engraft cells to sites such as kidney or testis capsule, subcutaneous or intramuscular sites. Subsequent teratoma formation is then assayed by staining of histological slides.
This technique can also be used to transfer small amounts cells or tissue such as thymic lobes to study their in vivo development into differentiated tissue.
Image: teratoma with a big skin structure, generated by iPS cells.
- Recipient animals including NOD/SCID and isolator space
- Screening of cells for pathogens prior to transfer
- Grafting of cells or tissues to an approximate site for teratoma or tissue formation
- Dissection and processing of tissue for histological staining or immunohistochemistry, using paraffin or cryostat sections as required
- Histological scoring and a written report on the subsequent quality and differentiation of the tissue including photomicroscopy