Hi everyone, I would like to ask if I can transfect MDA-MB231 breast cancer cells with 2 different DNA plasmids. I mean different genes. Is it possible?
thank you
I think you can. Because this is what people do for a dual luciferase reporter assay just as one example. However you should consider what expression level you expect from both genes. (I assume you are planning a transient expression and not a stable transformation. In this case you must select many individual cell clones to prove that both transgenes are indeed integrated into the genome.) It might be necessary to adjust the relative amounts of the two plasmids to obtain a useful result, especially when the two gene products interact. An alternative would be to construct an IRES vector encoding both proteins on a single transcript.
You certainly can. Several approaches, which use internal controls (as mentioned by Norbert above), transfect two or more plasmids at the same time; also of note, several approaches transfect libraries of plasmids. As a rule of thumb individual mammalian or yeast cells will take up as many plasmids you give them, essentially different from competent bacteria which will take only a single plasmid (the basis of cloning).
As Norbert mentioned, you should pay attention to the expression levels. Two things might influence the absolute and relative expression (in addition to respecting the molar equivalent of the transfected plasmids - a plasmid that is twice the size will have half as many copies in your transfection if you add the same mass in micrograms, for example):
1. Plasmids that contain an SV40 origin of replication (in the plasmid map shown as SV40 ori) will achieve very high numbers in 293T cells (or any cell that expresses T antigens - which drive the plasmid replication). If you have one plasmid with and one without, you might see wildly different levels for both plasmids which will be reflected in levels as well.
2. If both plasmids share the same promoter (say, CMV) you should expect a phenomenon called 'squelching' in which plasmid promoters compete for a TF and one influences the expression of the other.
Hi Hamad,
it is possible but mda-mb231 cells are hard to transfect with most commercial lipid-based reagents. Thus, the fraction of cells bearing both plasmids will be very low, usually allowing only single cell analyses (e.g. IF).
hope this helps,
Metello
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