Transgenesis
The Transgenesis Unit (TU) has, as main objective, the production of genetically engineered mice by artificially introducing foreign DNA molecules into their genomes (the so called transgenic mice) and supply them, as required, to the different CNIC scientific groups in accordance with their research need. From a biomedical point of view, such mice represent a tool of paramount importance to study and understand the gene function in vivo within the complexity of a whole organism. This has led to the development of animal models of human diseases allowing improved understanding of the diseases and, in several cases, to the development of new and more efficient therapies. In our case, they will serve as starting point to analyze basic processes of the cardiovascular physiology and homeostasis as well as to assay new experimental therapies.
Other important goals of the TU are:
- To rederivate mice strains into the sterile area of the Experimental Unit
- To cryopreserve the valuable produced and others mice strains
- To set up into our facilities the new reported techniques related with the reproductive and developing biology of the mouse
- To produce disease animal models by using surgical techniques
- To provide the reported and also the newest mouse transgenesis techniques as well as to develop new own technology in accordance with the CNIC research groups need
To achieve successfully and efficiently all of these goals, the unit has forefront equipment and appropriate facilities. Among others, the unit has two laboratories placed at the Experimental Unit: one is the so called micromanipulation/microinjection laboratory and the other is the laboratory for embryo transfers.
Among the techniques used at the TU to carry out its activities are:
- Production of transgenic mice by:
- Pronuclear Microinjection (the direct microinjection of DNA molecules into the pronuclei of fertilized oocytes)
- Subzonal o Perivitelline Microinjection of fertilized oocytes with genetically engineeered retrovirus/lentivirus
- Pronuclear and cytoplasmic microinjection in zygotes and 2-cell embryos with CrispR-Cas9 for the generation of transgenic mice
- Electroporation of mouse zygotes with CrispR-Cas9 for the generation of transgenic mice
- Production of chimeric mice to obtain Knock-Out (KO) and Knock-In (KI) mice by:
- Aggregation of eight-cell embryos (E8C) with genetically modified embryonic stem cells (ES-Cells)
- Microinjection of eight-cell embryos (E8C)/blastocysts with genetically modified embryonic stem cells (ES-Cells)
- Pronuclear and cytoplasmic microinjection in zygotes and 2-cell embryos with CrispR-Cas9 for the generation of KO / KI by floxing of regions or inserting cassettes.
- Rederivation of mouse strains by embryo transfer
- Cryopreservation of mouse strains by:
- Embryo freezing
- Sperm freezing
- In vitro Fertilization (IVF)
- Intracytoplasmic Sperm Injection (ICSI)
- Other techniques developed by the Transgenesis Unit and involved in the handling of experimental animals:
- Ovariectomy
- Ovarian transplant
- Artificial insemination techniques: intrabursal, transoviductal and intrauterine
- Techniques developed by the Transgenesis Unit and at the service of the CNIC
- Management of tetraploids (obtaining, aggregations and microinjection)
- Enucleation / Electrofusion techniques with karyoplasts and cytoplasts for the generation of conplastic mouse lines
- Cryopreservation Techniques for Zebrafish germplasm: sperm
