cotransfection selection plasmid - need antibiotic marker rather than B galactocidase (Dec/04/2008 )

hi all,

i am having trouble with a cell line we have just established via plasmid transfection

We had some good luck and managed the immortalisation pretty quickly and as the plasmid had a neomycin selection marker contained within it we could easily selct our immortalised cells with neomycin exposure in the cell medium.

Hence any cells which took up the plasmid would be resistant and would not die after exposure any cells which did not would die and we are left with a pure culture of immortalised cells

The problem we are having now is we have to do a further transfection with this cell line to insert a luciferase gene and this luciferase plasmid does not contain a selection marker, therfore we have to do a co-transfection with a plasmid containing a selection marker.

we are currently using B galactocidase however if both plasmids insert into the genome the only way we can find out is firstly by fixing (killing) the cells and then we still only get a colour change, which only gives us an indication as to how many cells have taken up the plasmids, and still does not seperate them out which i think would take a long time of subculturing small clusters of cells, unlike our first transfection which allowed us to select on antibiotic resistance

Anyway sorry for the long explanation but this gets me to my question,

Does anybody know of a plasmid which contains antibiotic resistance only, which we could use to co-transfect the cells instead of B galactocidase or indeed any plasmid that has only a selection marker as we really do not want to co-transfect with another plasmid containing antibiotic reistance (and other genes) aswell as our luciferase gene as we are trying to keep our cells as close to primary cell characteristics as possible

Many thanks for your help

Colm.

Hi,

I'd say the best thing would be to construct a single plasmid, containing both the luciferase gene and the selection marker. With co-transfection, unless both plasmids have different resistance to antibiotics, you can never be assured that your cells have taken up the 2 plasmids.

You could also make a viral construct, with both selection marker and your luciferase construction.