Aggregation of ES Cells and Eight Cell Stage Embryos
The aggregation method for generating chimaeras, as opposed to the microinjection technique, is useful as it does not require expensive microinjection apparatus or sophisticated manipulative skills, and utilizes outbred blastocysts which are easier to obtain in numbers. The ES cells are intorduced into the developing embryo by the adherance of the cells to a dezonulated 8 cell stage embryo. The embryos are cultured to blastocysts and then transfered into a pseudopregnant hybrid (C57 X CBA) F1 recipient.
Preparation of the aggregation plate.
Aggregation and culture of the embryos with the ES cells is performed in microwells prepared in a plastic tissue culture plate using a darning needle.
Removal of the Zona pellucida from the embryo.
Siliconizing Capillary Glass
Embryos that have had the zona pellucida (jelly-like outer coating) removed are "sticky" to handle, often getting stuck inside the transfer pipette during handling. This problem is overcome by siliconizing the capillary glass (inside only) before it is used to fashion transfer pipettes.
NOTE: This procedure must be carried out wearing gloves in a fume hood!
Use a 27 gauge needle and 3 cc syringe (filled) to run a small amount of silicone (Sigmacote) through the inside of a piece of capillary glass, allowing excess Sigmacote to drip into a 10ml tube. This is repeated with each piece of glass and the glass placed in a measuring cylinder. The excess Sigmacote in the 10ml tube may be used to refill the syringe when empty. Once 50-80 pieces have been siliconized the glass is rinsed no less than eight times with Milli-Q water, shaking the water out of the glass by hand after each rinse. The glass is then rinsed six times with "Travenol" water. The cylinder is then sealed with Parafilm which is punctured several times with a needle to allow evaporation with minimal dust collection. The cylinder is placed in a hot air oven to speed evaporation. Once completely dry, the glass is ready to use.
Sigmacote is made by Sigma Chemicals. The catalogue number for Sigmacote is SL-2
Preparation of ES cells for aggregation
We use small clumps of ES cells rather than single ES cells as these are much easier to place in the wells. The cells are harvested essentially as described in "Passaging and Spliting ES cells" except that at the end of the tyripsinization step they are GENTLY pipetted up and down 6-8 times rather than the vigourous motion usually used to create a single cell suspension. This yields clumps of cells of 4-12.
Removal of Expanded Blastocysts from Aggregation Wells
Before attempting to remove expanded blastocysts, make sure that the pipette is wide enough. To remove blasts from wells, fill a transfer pipette with M16 up to the end of the narrow pulled section then position the end over each well and blow the embryos out. Arrange them in groups of 12 for easy collection during transfer.
|Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: firstname.lastname@example.org |
David Bowtell PMCI October 1998