By Angel Cid-Arregui, Alejandro Garcia-Carranca
The means of microinjection is used to tranfer organic fabrics - RNA, proteins, oligonucleotides and particularly cloned genes - into cultured somatic cells or embryos. Transgenic cells or animals - mice, rabbits or bugs - let the examine of a variety of mobile procedures, corresponding to gene expression, sign transduction or the cytoskeleton. greater than 30 protocols on microinjection experiments and transgenesis reviews built in popular laboratories are defined during this handbook. particular subject matters are the new release of transgenic mice utilizing YAC, ligand-dependent site-specific recombination in embryonic stem cells, morula aggregation to generate germline chimeras in addition to the research of injected cells.
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Extra info for Microinjection and Transgenesis: Strategies and Protocols
3, upper left panel). Manual intranuclear injection 22 ANGEL CID-ARREGUI ET AL. 3. Apply injection pressure: press on the footpath switch of the microinjector to apply P2 for the selected time (see previous section). If a syringe is being used, ensure that enough pressure is applied at this moment. 4. Move the micropipette upwards carefully until it leaves the cell. 5. Move the microscope stage to find a new cell, center its nucleus with respect to the tip and repeat steps 2 to 4. If Cellocate coverslips are used (see Materials, above), injected cells can be recorded in the book provided by the manufacturer.
Incubate the injected cells for the appropriate time. 2. Rinse in PBS, drain. 3. Scrape the cells with the tip of a pipette loaded with 100 III of TGD buffer (250 mM Tris pH 8, 15 % glycerol, 5 mM dithiotreitol) while pipetting up and down. Transfer cell suspension to a lo5-ml microfuge tube. 4. Prepare crude extracts either by sonicating in a water bath or by performing five to six freeze-thaw cycles in a dry-ice/ethanol bath and a 37°C water bath. 5. Centrifuge at 4°C for 10 min to eliminate debris.
Fix capillary in the holder and mount it on the micromanipulator. 6. Focus on cells and bring the capillary into the field of observation as described above. 7. Do some test injections to select proper injection pressure and injection time. 8. Switch to automatic mode. 9. Inject 100 cells into the cytoplasm with the FITC solution. 2 Automated Computer-Assisted Microinjection into cultured somatic cells 41 10. Then, restart and remove petri dish, put back into incubator, incubate for 1 h. Well-injected cells will survive, cells damaged too much by the microinjection will die and usually detach from the coverslip 11.
Microinjection and Transgenesis: Strategies and Protocols by Angel Cid-Arregui, Alejandro Garcia-Carranca