Preparing ES Cells for Injection
Our lab has injected a large number of constructs from a variety of labs and with several different ES cell lines. We have noticed that the two most important factors that affect chimerism rates are morphology and confluency. The colonies should be small, tight, have smooth raised edges, and in log phase growth. Confluence should be ~60-70%, with many small to medium size colonies. The ES cells are considered overgrown when they are no longer individual colonies and have begun to run into each other. It is important to have the cells in log phase growth because we are looking for the ES cells to overtake the embryo. When the ES cells are overgrown and have plateaued the ES cells do not contribute as large a percentage to the embryo. The result is lower numbers of chimeric animals and lower percentage of chimerism in the individual founders with reduced germline transmission.
Because different cell lines and clones grow at different rates it is best to prepare a least two dilutions of each clone for injection. A simple method is to thaw your vial of cells for injection, or passage the clone, 1/3 in one well and 2/3 in the second well. If you are low in vial numbers for each clone we will be happy to return any unused wells back to you, otherwise we discard any cells that we do not use.
When you are ready for injection we would prefer at least two dilutions of each clone, with each dilution growing in one well of a 6-well plate. To change the smallest number of variables we ask for an aliquot of your ES cell media (NO DRUGS) and trypsin. We use that media to inject the ES cells into the embryos. Feed the ES cells 2-3 hours before injection (8-10 AM) to make sure they are in log phase growth for injection and bring them to out lab by 11:00. We inject the ES cells ~ 12 AM in the morning Tuesday - Friday. We inject up to 4 clones per paid construct.