Isogenic human cell lines for drug discovery: regulation of target gene expression by engineered zinc-finger protein transcription factors.
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.
Liu
Pei-Qi
PQ
Sangamo BioSciences, Inc., Richmond, CA 94804, USA.
Tan
Siyuan
S
Mendel
Matthew C
MC
Murrills
Richard J
RJ
Bhat
Bheem M
BM
Schlag
Brian
B
Samuel
Rachelle
R
Matteo
Jeanne J
JJ
de la Rosa
Ragan
R
Howes
Katherine
K
Reik
Andreas
A
Case
Casey C
CC
Bex
Frederick J
FJ
Young
Kathleen
K
Gregory
Philip D
PD
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
D017931:DNA Primers; D012333:RNA, Messenger; D044168:Receptor, Parathyroid Hormone, Type 1; D014157:Transcription Factors