contransfection why choose beta galactocidase? - why not an antibiotic resistant plasmid (Dec/12/2008 )
Can anybody tell me why people still use beta galactocidase as a positive control when performing contransfection with another plasmid
The reason i ask is that we are trying to transfect a luciferase plasmid (with no selection marker) into our recently immortalised cells
As most people seemed to use beta gal we also tried this but when trying to work out if a cell has taken up both plasmids we first have to kill the cells and the results only show a percentage of how many cells have taken up the plasmids and we are still left with a mixed population of cells which requires a lot of subculturing etc to isolate specific cells with beta gal plasmid
Can anybody tell me why people dont use an antibiotic plasmid with no other insert (such as pBABE-puro www.addgene.org/1764) for selection instead?
Surely this is a more efficeint technique as non transformed cells will die on exposure to puromycin and removes subculturing etc?
Maybe i am wrong and this way is what most people are doing already but if it is i have not seen it mentioned in any recent papers
Is this beacause most plasmids would have selection markers already inserted and so positive controls are not needed?
Or is their some other reason such as beta gal being pretty kind to the cells in terms of differentaiation than an antibiotic resistant plasmid would be?
Any help/advice/suggestions would be great,
Sorry, it's friday, I'm not sure I understood well, but it looks like you want to do stable transfection and kill all cells that do not have been transfected. This is performed by using only one plasmid containing your gene of interest and an antibiotic resistance gene.
co-transfection is used for transient transfection control.
you are kind of correct, yes i want to do a transfection with a luciferase plasmid which we have, but the problem is this luciferase plasmid does not contain a selection marker so we have no idea if this has been taken up by the cells.
therefore we have to do a co-transfection at the same time so the cells will hopefully take up the two plasmids at once, one being our luciferase plasmid and the other being the selection plasmid (as i explained normally beta galactocidase but i want to use an antibiotic resistant one instead) so we can then treat the cells with the antibiotic to select for our cells
does this make sense to you?
basically i wanted to know why people continue to use beta galactocidase if it is easier to use an antibiotic plasmid for co-transfection
in my opinion it doesn't make sense ( but I'm not a specialist of co-transfection) because you will select the cells that have the marker plasmid, but there is no evidence that these cells have also kept the luciferase plasmid.
In the experiment I'm planning to perform with co-transfection, I will use one plasmid to transduce shRNA and one plasmid to transduce the target of shRNA together with reporters to control the efficiency fo the shRNA. it would be transient transfection. The goal is not to label the transfected cells here.
yes i agree in principal it does not make sense but the person who designed this particular plasmid for luciferase did not incorporate an antibiotic resistance into it
alot of people also perform this exact method using beta gal whichs give very little selection, depending on the ratio of Luciferase:antibiotic plasmid papers and manufacturers reckon that you can get anywhere from 10 - 100 % of cells taking up both plasmids at the same time
so then after treatment with the antibiotic we still have to establish if these clones have the luciferase but at least we can trypsinise clones seperately and start a new culture from each, then if we find out one of the clones has luciferase gene we will have a separate flask of cells from them and can establish many more passages from it
i know it sounds very complicated but far better than using the beta gal as most people who need to co-transfect due to no antibiotic marker on their plasmid have to do
sure I agree that selection with an antibiotic is far better than using beta gal, but may be someone using beta gal coud tell us why we would be wrong ?
Nobody is using beta gal for this purpose here ?