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Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells
Authors:Brigham J Hartley  Stewart A Fabb  Ben AL Finnin  John M Haynes  Colin W Pouton
Institution:1.Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University
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.
Keywords:Molecular Biology  Issue 90  Electroporation  human embryonic stem cell  genome editing  reporter cell line  midbrain dopaminergic neurons
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