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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?

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Depending on the stage of cell-cycle you are interested in, people often use the size/morphology of the cell as an indicator. Then you select cells with a cell sorter.

Another option is to specifically tag some cellular molecule that can give an indication as to the relevant stage and then sort the cells based on the tag. This does not necessarily have to be cyclins - for example if you are only interested in S/G2 you could tag the DNA or maybe histones.

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You have correctly stated that we can monitor cell cycle stage by either monitoring protein (cyclin) levels or by following indirect methods such as green cassettes. One usually tries to monitor changing levels of:

  • G1 cyclins (D cyclins)
  • S-phase cyclins (cyclins E and A)
  • Mitotic cyclins (B cyclins)

Or one might try to modify the gene where these proteins are produced so that one can observe some unique behavior that highlights that particular stage of cell cycle is going on.

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Thanks for the info! Though it doesn't quite answer what I wanted to know - I added more detail to my question as I can understand that I was a bit unclear. – Armatus Jan 6 '13 at 16:49

The only strategy close to your suggested description is based on FUCCI sensors. Fluorescent markers designed so that their presence informs of specific stages of the cell cycle, in principle without disrupting or perturbing cell progression.

So, although there is not a physical clean-up of pure cell sub-populations, these can be tracked by the abundance of these markers and even isolated by FACS.

I guess the issue of developing something selectively "lethal" (which does exist in the context of certain plasmids transformed in E.coli) lies partly in being able to retrieve those surviving cells in a timely fashion so that being halted at that particular stage is not detrimental. It does sound as an interesting analogy to Sanger Sequencing where 4 different terminators of each of the 4 bases render truncated fragments easily readable. In principle within the context you suggest, a single cycle would be allowed post genetic manipulation (eventual silencing of such genetic modification as representing an undesirable weight for the cell would be expected otherwise). As progression through each phase varies substantially in time, the idea in itself would require substantial refinement, and most likely the use of a conditional expression system. In this way a mix population transfected with the specific set of markers could be triggered for expression at a given time and then monitored for negative selection.

Commonly though, chemical synchronisation or perhaps more physiologically relevant methods of separation like elutriation, aim at enriching cell populations at specific stages of cell division. Mitotic cells are particularly problematic being rather scarce at any given time within an unperturbed population of growing cells. Mitotic shake-off followed by addition of hypotonic solutions have been suggested as methods of mitotic retrieval but yield is comprehensibly low.

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