title: Isogenic human cell lines for drug discovery: regulation of target gene expression by engineered zinc-finger protein transcription factors.
abstract: 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.
authors.affiliation: Sangamo BioSciences, Inc., Richmond, CA 94804, USA.
authors.forename: Pei-Qi
authors.initials: PQ
authors.lastname: Liu
authors.forename: Siyuan
authors.initials: S
authors.lastname: Tan
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Mendel
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Murrills
authors.forename: Bheem M
authors.initials: BM
authors.lastname: Bhat
authors.forename: Brian
authors.initials: B
authors.lastname: Schlag
authors.forename: Rachelle
authors.initials: R
authors.lastname: Samuel
authors.forename: Jeanne J
authors.initials: JJ
authors.lastname: Matteo
authors.forename: Ragan
authors.initials: R
authors.lastname: de la Rosa
authors.forename: Katherine
authors.initials: K
authors.lastname: Howes
authors.forename: Andreas
authors.initials: A
authors.lastname: Reik
authors.forename: Casey C
authors.initials: CC
authors.lastname: Case
authors.forename: Frederick J
authors.initials: FJ
authors.lastname: Bex
authors.forename: Kathleen
authors.initials: K
authors.lastname: Young
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D015202:Protein Engineering; D012333:RNA, Messenger / Q000235:genetics; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000737:chemistry / Q000502:physiology*; D016335:Zinc Fingers
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger; D044168:Receptor, Parathyroid Hormone, Type 1; D014157:Transcription Factors