Proliferation and fate Flashcards
What is quiescence?
Quiescence - temporary persistence in the G0 phase of the cell cycle. Most stem cells are quiescent especially when compared to their direct progeny. Note: differentiated cells are not quiescent; they are post mitotic and cannot proliferate anymore.
What are the two kinds of G0 phase?
There two kinds of G0
- quiescent - the one stem cells use
- post mitotic state - cells that are in this stage are out of the cell cycle and are never going back.
How can we check how much proliferation we have?
To answer these questions we can check for markers such as Ki67 and BrdU
What is BrdU?
BrdU is an analogue of thymidine and gets incorporated in stems that are in S phase. So when we use BrdU we are specifically labelling cells that are duplicating their DNA.
How can we see which cells are in particular phases?
We can see which cells are in particular phases. We can use a dye such as DAPI or Hoechst to label the cells (because they are fixed the dye will go in and not be pumped out).. Then we can use flow cytometry on the cell suspension and get the plot. The more dye there is in the cell the more DNA is present.
What chemicals can also be used to characterise cells based on the stage of the cell cycle they are in?
We can also use different cyclins as markers as they are expressed at different stages of the cell cycle
What is a FUCCI reporter system?
FUCCI reporter system - fluorescent, ubiquitination based cell cycle indicator
What phase does Cdt1 indicate?
Cdt1-KuOrange in G1
What phase does Geminin indicate?
Geminin-GFP in S, G2 and M
Describe a label retaining cell assay
- Short term administration of the lable highlights all cells
- Afterwards dividing cells dilute the lable between daughters.
- Non-dividing cells retain the lable and are detected as label retaining cells
What happens during the chase phase and why is it important?
during the chase phase proliferation happens. The more a cell proliferates the less dye remains in the cell → cells that don’t proliferate much (such as stem cells) remain strongly labeled.
What are some limitations of BrdU?
- BrdU
- you need to fix cells to use BrdU so you cannot perform functional assays afterwards and can’t really tell if the cell is the stem cell
- is slightly toxic so some cells die which the activates stem cells
- you need to have dividing cells so you need to perform the staining at a young age of the mice. If the stem cell s in quiescence then they won’t be labeled
How does the Tet-regulated H2BGFP system work?
The Tet gene expression system functions when a recombinant tetracycline-controlled transcription factor (either tTA for Tet-Off or rtTA for Tet-On) binds to theTet-op promoter, subsequently driving the expression or controlling the inhibition of the target gene (see figure below).
Gene expression is regulated by the presence or absence of tetracycline or one of its derivatives such as doxycycline. Tetracycline binds directly to the transcription factors.
Tet-Off system: doxi binds the tTa and takes it away from the promoter turning the system off.
Tet-On system: doxi binds the rtTa and allows it to bind to the promoter turning the sysetm on.
Which cell will remain bright in the Tet-regulated H2BGFP system?
only the cells that do not proliferate remain bright
Which cells are marked in an ubiquitous system?
- in the example above we use an ubiquitous system which allows us to monitor cells everywhere in the mouse which might be nuce as we can then see the dilution of fluorescence in different tissues
- we can also use tissues specific systems to only monitor the events in particular tissues