Cell cycle selection?

Question

Is it possible to select from colonies only cells which are at a certain stage in the cell cycle? E.g. if I was trying to analyse expression of a number of genes during different stages of the cell cycle.I could imagine that if this is possible, the selection mechanism might target cyclins, or use an artificial gene cassette controlled by them.So for example, a method may attach some gene to a certain cyclin which protects against a substance that otherwise destroys the cell. We could then use that substance to destroy all cells which were not in the cell cycle stage that we want to look at, wash away their nucleic acids, and then extract the mRNA from remaining cells to analyse expression levels at this particular stage in the cell cycle. If we repeat that for the different stages, we should be able to get a nice picture of what is expressed when. Does any method like this exist or is commonly used?

Answer 1

Yes, for cells growing in a culture dish, you can use several different drugs that prevent the cells from progressing through a specific phase of the cell cycle (eg nocodazole for M phase, aphidicolin or mimosine for S phase). This will cause them to halt at that phase so that over time the population will be "synchronized". When the drug is removed, the cells will resume their transit through the cell cycle as a synchronized population. However, the drawback is the toxicity or other cellular effects of the drug used for synchronization.

You can also use a non-drug method called a “mitotic shake off”. When cultured cells are in M phase, they round up and are loosely adhered to the culture dish. You can harvest these cells by gently washing them off the dish with culture medium. Tilt the dish slightly and very gently pipette the culture medium from the dish over the cells several times. After 5-6 times, the culture medium will have M phase cells, which can then be re-plated and analyzed as they divide and enter G1 phase. However, since M phase is very short, only about 5% of the cells in an asynchronously growing population will be in this phase and the % yield will be small. You may have to use many large (10 cm) dishes, or collect the mitotic cells at several successive time points. You can also increase the yield by combining the shake off method with the drug nocodazole, which will halt cells in M phase, so that a larger number of cells can be harvested with the shake-off.

Here are some references on synchronization methods:

Jackman J, O'Connor PM. Methods for synchronizing cells at specific stages of the cell cycle. Curr Protoc Cell Biol. 2001 May; Chapter 8:Unit 8.3. doi: 10.1002/0471143030.cb0803s00.

https://www.oxfordreference.com/view/10.1093/oi/authority.20110803100202189

Schorl C, Sedivy JM. Analysis of cell cycle phases and progression in cultured mammalian cells. Methods. 2007 Feb;41(2):143-50.