Selection of StableTransfectants Expressing a High Density of Cloned M - (Jul/25/2001 )
Hello....We are trying to establish several stably transfected cell lines using CHO and HEK cells transfected with vectors containing our cloned, membrane-expressed receptors. After selecting for transfected cells with the appropriate antibiotic, we are left with a population of transfected cells, each potentially expressing very different levels of our cloned receptor. To select for cells with the highest level of expression, we can perform ligand binding assays to determine the levels of expression in different cells. However, should we be trying to select a SINGLE transfected cell expressing a high density of receptors on its membrane and grow up more cells from that single cell? If so, how do I single out one cell - by plating out my transfected cells at a very low dilution to try and get one cell per well, and then testing each well (by our ligand binding assay) for receptor density? Or is this unneccessary, i.e. is it OK to just select a heterogeneous population of cells that are expressing a high density of our cloned receptor on their surface? Later studies we wish to perform on these cells are ligand binding assays, receptor trafficking sudies, and receptor-receptor interactions. Any advice on what I should be doing would be GREATLY appreciated.Thanks,Alljson
Hi. We are trying to overexpress a membrane bound receptor also. What sort of ligand binding assay to you perform and how does it work? Thank you.