Hello RG community,
I wonderer whether exogenous molecules (e.g. reporter plasmids, miRNAs or other artificial molecules) are transferred between cultured human cells during their doubling ?
So, here's my question. If I seed 50 k cells with a doubling time of 24 h (Hek293) in a 12-well plate on day 1 and transfect them on day 2, practically 100 k cells should be transfected with my molecules of interest. Eventually, I leave the cells for 3 more days in the incubator before I disintegrate the cells to perform my final assay (e.g. Luciferase assay). Is it so that I measure luciferase activity from the 100 k cells that I transfected or are there potentially more than 100 k cells been carrying my transfected molecules?
Can anyone give me a hint of how many cells might eventually be involved ?
Thanks for your comments!
Hi, Firstly, not all 50k cells will get transfected. How many cells (%) will get transfected (the efficiency of transfection) depends on many factors.
For example,
- the transfection method you choose
- how clean the DNA and the ratio of the DNA and transfection reagent
- how healthy the cells are
- whether you have optimized the protocol, how much to add, how long to keep.
If you want to see how many cells got transfected, use a GFP expressing vector, and observe it in immunofluorescent scope. will have an idea.
Exogenous molecules are most definitely transferred to daughter cells during mitosis. For example, I've used a self-replicating mRNA that persisted under antibiotic selection in HEK293T cells for 3 months (We stopped the experiment at that point but had not observed any indication of decreased expression from the mRNAs).
You can easily observe this phenomenon using GFP expressing plasmids at a low transfection. At 24 hours after transfection, you'll see a large number of the fluorescent cells are present as pairs within the monolayer.
When doing these sorts of experiments in cell cultures it is very important to plate all of your wells/plates from a single suspension of your cells. While this won't guarantee a perfectly equal number of cells in each well/plate, it will definitely reduce the variability. You can further reduce variability by performing technical replicates within a single experiment (i.e. transfect 3 wells with each of your exogenous molecules).
@Chandra Somasundaram: Thanks for your response. I haven't thought about such factors. Will consider it.
@John Hardy Lockhart: Thanks for your response and the useful suggestions. Will plan accordingly.
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