Week 5

                This week was spent drug selecting the cells. The plasmid that was inserted into the cells contains a gene for resistance of a drug called puromycin. Drug resistance is important in selecting which cells contain a certain plasmid, because electroporation is not nearly one hundred percent effective. Only a few cells take up the plasmid, and we need an effective method for isolating these cells from all the others. Drug selection is a useful tool in this process because all cells that do not contain the resistance gene die off, leaving only the cells containing the plasmid to proliferate and form colonies.

                Maintaining a good density of cells on the plate is very important when attempting to create a stable cell line. The confluence of the cells on the plate should not exceed 80 percent. This is because cells respond to a form of inhibition called density dependent inhibition. This means that cells can only divide when there is room for them on the surface they are attached to; when they run into each other, they stop dividing and go into quiescence. If the plate becomes too confluent, the cells will undergo apoptosis and die. Therefore, when maintaining a cell culture, it is important to make sure the plates are split regularly enough that the plate does not become too confluent.