NDLI: Zinc-finger nuclease enhanced gene targeting in human embryonic stem cells.

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Zinc-finger nuclease enhanced gene targeting in human embryonic stem cells.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Hartley, Brigham J. Fabb, Stewart A. Finnin, Ben A. L. Haynes, John M. Pouton, Colin W.
Description	Author Affiliation: Hartley BJ ( Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University.); Fabb SA ( Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University.); Finnin BA ( Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University.); Haynes JM ( Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University.); Pouton CW ( 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.
File Format	HTM / HTML
e-ISSN	1940087X
DOI	10.3791/51764
Journal	Journal of Visualized Experiments
Issue Number	90
Language	English
Publisher	MyJove Corp.
Publisher Date	2014-08-23
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Deoxyribonucleases Metabolism Embryonic Stem Cells Physiology Gene Targeting Zinc Fingers Animals Cell Tracking Dna Breaks, Double-stranded Chemistry Electroporation Cytology Homeodomain Proteins Genetics Mice Recombinant Fusion Proteins Transcription Factors Research Support, Non-u.s. Gov't Video-audio Media
Content Type	Text
Resource Type	Article

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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