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Isogenic Human Cell Lines for Drug Discovery: Regulation of Target Gene Expression by Engineered Zinc-Finger Protein Transcription Factors

Siyuan Tan

Matthew C. Mendel

Sangamo BioSciences, Inc., Richmond, CA

Richard J. Murrills

Bheem M. Bhat

Women’s Health & Bone, Wyeth Research, Collegeville, PA

Brian Schlag

Neurosciences, Wyeth Research, Princeton, NJ

Rachelle Samuel

Jeanne J. Matteo

Women’s Health & Bone, Wyeth Research, Collegeville, PA

Ragan de la Rosa

Katherine Howes

Andreas Reik

Casey C. Case

Sangamo BioSciences, Inc., Richmond, CA

Frederick J. Bex

Women’s Health & Bone, Wyeth Research, Collegeville, PA

Kathleen Young

Neurosciences, Wyeth Research, Princeton, NJ

Philip D. Gregory

Sangamo BioSciences, Inc., Richmond, CA

Isogenic cell lines differing only in the expression of the protein of interest provide the ideal platform for cell-based screening. However, related natural lines differentially expressing the therapeutic target of choice are rare. Here the authors report a strategy for drug screening employing isogenic human cell lines in which the expression of the target protein is regulated by a gene-specific engineered zinc-finger protein (ZFP) transcription factor (TF). To demonstrate this approach, a ZFP TF activator of the human parathyroid hormone receptor 1 (PTHR1) gene was identified and introduced into HEK293 cells (negative for PTHR1). Following induction of ZFP TF expression, this cell line produced functional PTHR1 protein, resulting in a robust and ligand-specific cyclic adenosine monophosphate (cAMP) response. Reciprocally, the natural expression of PTHR1 observed in SAOS2 cells was dramatically reduced by the introduction of the appropriate PTHR1-specific ZFP TF repressor. Moreover, this ZFP-driven PTHR1 repression selectively eliminated the functional cAMP response invoked by known ligands of PTHR1. These data establish ZFP TF–generated isogenic lines as a general approach for the identification of therapeutic agents specific for the target gene of interest.

Key Words: zinc-finger protein • high-throughput screening • G protein–coupled receptors • isogenic cell lines

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