Markers and selection in transfection? - (Jan/04/2007 )
I did trnsfections a couple of times and still have silly questions about this. What are markers? And how are they linked to selection pressure applied after transfections?
My understanding: Markers are the within the gene inserts used for transfection. And to see if the gene of interest is properly inserted into the cells after transfection or not, we apply selection pressure - either by specific antibiotics like hygromycin / neomycin or others. Then the cells that are not killed and that which survive the selection process, are the ones that are successfully transfected. Am I right in this explanation? Then isn't it true that the selection agents should be selected only after deciding the gene of interest? - the gene to be inserted? because the marker should be present within this region - Right? Or Is it true that the markers are present in the particular cells like HEk 293 or HeLa cells already and that the selection antibiotics to be used are pre-decided for these cells, irrespective of the gene to be inserted / transfected. Please help me understand better.
Thanks for your replies in advance.
There are severall kinds of markers. There are the detectable markers like fluorescent proteins, luciferases etc, that will enable you to detect expression by fluorescence microscopy/facs/fluoreometry or by measuring RLU's for luciferases. When cloned next to a certain promoter, you can check if that promoter is active in you cell of interest, wether or not expression is increased/decreased by some kind of treatment.
Second kind of marker is a selectable marker, like the gene encoding for neomycin resistance. Mostly, these genes are included in a plasmid with a constitutive promoter so that you can select transfected cells. The plasmid also will contain a gene or another DNA sequence that you want to do your research on. When you select a stable transfectant by antibiotic pressure, you have a high chance that these cells will also express your gene of interest (or have your DNA sequence in the cell).