Lynn H. Caporale

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.

Parathyroid Hormone Antagonists Effective in Vivo

There has long been great impetus for preparing peptide hormone antagonists effective in vivo. Pure hormone antagonists can be used to determine the mechanism of action of peptide hormones and to elucidate both the role of a hormone in normal physiology and its contribution to pathophysiology. For parathyroid hormone in particular, the impetus for generating antagonists effective in vivo includes a wide variety of therapeutic and diagnostic clinical uses in hyperparathyroid conditions (Table 1) and diagnostic utility in disorders of calcium metabolism.

New Directions for the Design of Parathyroid Hormone Antagonists

Peptide hormone antagonists that are effective in vivo are uniquely precise tools for biomedical research. They can be used to determine how peptide hormones act, what their role is in normal physiological processes, and how they contribute to pathophysiologic states.

Gene transfer by protoplast fusion: Expression cloning of rare gene products in mammalian cells

A method is described that facilitates performing a large number of protoplast fusions to mammalian cells simultaneously and successfully. This method makes it possible to circumvent typical hurdles to the use of transient expression in mammalian cells, facilitating expression cloning of DNA enoding the newly detected gene product of interest. The original in vitro assay used to define the new activity is of interest, adapted to microtiter plates, combined with protoplast fusion, extends the reach of expression cloning to such cases as products of a rare message, or activities involving a multisubunit or unstable protein or both.