Protoplast fusion in microtiter plates for expression cloning in mammalian cells: demonstration of feasibility using membrane-bound alkaline phosphatase as a reporter enzyme.
Protoplast fusion is a method for directly transferring cloned DNA from bacteria to mammalian cells at high efficiency. Here, we have used membrane-bound alkaline phosphatase as a reporter enzyme in a miniprotoplast fusion assay. This work demonstrates the principle that large numbers of protoplast fusions can be done simultaneously and successfully, to assay for an activity encoded by an expression vector. The technique described here circumvents key hurdles to expression cloning. This method does not require a highly sensitive assay or a way of separating a rare expressing cell from the mixture of transfected cells containing other transfected genes. With a strong promoter, the protein encoded by the undiluted transfected cDNA should be produced at at least as high a level as it is endogenously produced in the cell from which its activity was first detected. Reference clones are stored, avoiding the need to separate out the cells that are successfully transfected; this also avoids the need to repurify the DNA from the transfected cell. Because of the use of microtiter plates, it is likely that such a method could be partially automated for many types of assays.
Caporale
L H
LH
Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Chartrain
N
N
Tocci
M
M
DeHaven
P
P
D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D007700:Kinetics; D011401:Promoter Regions, Genetic; D011523:Protoplasts / Q000378:metabolism*; D014162:Transfection
D000469:Alkaline Phosphatase