Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells
Autor: | John M. Haynes, Brigham J. Hartley, Stewart A. Fabb, Colin W. Pouton, Benjamin Arthur Llewellyn Finnin |
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Rok vydání: | 2014 |
Předmět: |
Homeobox protein NANOG
Recombinant Fusion Proteins Cellular differentiation General Chemical Engineering Biology Stem cell marker General Biochemistry Genetics and Molecular Biology Mice Animals Humans DNA Breaks Double-Stranded education Molecular Biology Cell potency Embryonic Stem Cells Homeodomain Proteins education.field_of_study Deoxyribonucleases General Immunology and Microbiology General Neuroscience Gene targeting Zinc Fingers Zinc finger nuclease Embryonic stem cell Molecular biology Cell biology Electroporation Cell Tracking Gene Targeting Pituitary homeobox 3 Transcription Factors |
Zdroj: | Journal of Visualized Experiments. |
ISSN: | 1940-087X |
Popis: | 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. |
Databáze: | OpenAIRE |
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