title: Zinc-finger nuclease enhanced gene targeting in human embryonic stem cells.
abstract: One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations. These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes. This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered. However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient. This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson's disease modeling or cell replacement therapy.
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University.
authors.forename: Brigham J
authors.initials: BJ
authors.lastname: Hartley
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University.
authors.forename: Stewart A
authors.initials: SA
authors.lastname: Fabb
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University.
authors.forename: Ben A L
authors.initials: BA
authors.lastname: Finnin
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University.
authors.forename: John M
authors.initials: JM
authors.lastname: Haynes
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University; colin.pouton@monash.edu.
authors.forename: Colin W
authors.initials: CW
authors.lastname: Pouton
mesh_terms: D000818:Animals; D058948:Cell Tracking; D053903:DNA Breaks, Double-Stranded; D003851:Deoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D018274:Electroporation / Q000379:methods; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D018390:Gene Targeting / Q000379:methods*; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D016335:Zinc Fingers
chemical_list: D018398:Homeodomain Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C433629:homeobox protein PITX3; D003851:Deoxyribonucleases