title: Genome editing of human embryonic stem cells and induced pluripotent stem cells with zinc finger nucleases for cellular imaging.
abstract: BACKGROUND
  Molecular imaging has proven to be a vital tool in the characterization of stem cell behavior in vivo. However, the integration of reporter genes has typically relied on random integration, a method that is associated with unwanted insertional mutagenesis and positional effects on transgene expression.
  
  
  OBJECTIVE
  To address this barrier, we used genome editing with zinc finger nuclease (ZFN) technology to integrate reporter genes into a safe harbor gene locus (PPP1R12C, also known as AAVS1) in the genome of human embryonic stem cells and human induced pluripotent stem cells for molecular imaging.
  
  
  RESULTS
  We used ZFN technology to integrate a construct containing monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase reporter genes driven by a constitutive ubiquitin promoter into a safe harbor locus for fluorescence imaging, bioluminescence imaging, and positron emission tomography imaging, respectively. High efficiency of ZFN-mediated targeted integration was achieved in both human embryonic stem cells and induced pluripotent stem cells. ZFN-edited cells maintained both pluripotency and long-term reporter gene expression. Functionally, we successfully tracked the survival of ZFN-edited human embryonic stem cells and their differentiated cardiomyocytes and endothelial cells in murine models, demonstrating the use of ZFN-edited cells for preclinical studies in regenerative medicine.
  
  
  CONCLUSIONS
  Our study demonstrates a novel application of ZFN technology to the targeted genetic engineering of human pluripotent stem cells and their progeny for molecular imaging in vitro and in vivo.
authors.affiliation: Department of Medicine, Division of Cardiology, Stanford School of Medicine, Stanford, CA, USA.
authors.forename: Yongming
authors.initials: Y
authors.lastname: Wang
authors.forename: Wendy Y
authors.initials: WY
authors.lastname: Zhang
authors.forename: Shijun
authors.initials: S
authors.lastname: Hu
authors.forename: Feng
authors.initials: F
authors.lastname: Lan
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Lee
authors.forename: Bruno
authors.initials: B
authors.lastname: Huber
authors.forename: Leszek
authors.initials: L
authors.lastname: Lisowski
authors.forename: Ping
authors.initials: P
authors.lastname: Liang
authors.forename: Mei
authors.initials: M
authors.lastname: Huang
authors.forename: Patricia E
authors.initials: PE
authors.lastname: de Almeida
authors.forename: Jong H
authors.initials: JH
authors.lastname: Won
authors.forename: Ning
authors.initials: N
authors.lastname: Sun
authors.forename: Robert C
authors.initials: RC
authors.lastname: Robbins
authors.forename: Mark A
authors.initials: MA
authors.lastname: Kay
authors.forename: Fyodor D
authors.initials: FD
authors.lastname: Urnov
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Wu
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D003851:Deoxyribonucleases / Q000008:administration & dosage / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000201:enzymology* / Q000637:transplantation; D018390:Gene Targeting / Q000379:methods; D017930:Genes, Reporter / Q000502:physiology; D005818:Genetic Engineering / Q000379:methods*; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000201:enzymology* / Q000637:transplantation; D051379:Mice; D061848:Optical Imaging / Q000379:methods; D017393:RNA Editing / Q000235:genetics*; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D003851:Deoxyribonucleases