Abstract |
Chronic granulomatous disease (CGD) is an immune deficiency characterized by defects in the production of microbicidal reactive oxygen species (ROS) by the phagocytic oxidase ( phox) enzyme complex in neutrophils. We have previously described targeted gene editing strategies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases ( TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nucleases for gene targeting with homology-directed repair in CGD patient stem cells to achieve functional restoration of expression of phox genes and NADPH oxidase activity in differentiated neutrophils. In this chapter, we describe detailed protocols for targeted gene editing in human-induced pluripotent stem cells and hematopoietic stem cells and for subsequent differentiation of these stem cells into mature neutrophils, as well as assays to characterize neutrophil identity and function including flow cytometry analysis of neutrophil surface markers, intracellular staining for phox proteins, and analysis of ROS generation.
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Authors | Colin L Sweeney, Randall K Merling, Suk See De Ravin, Uimook Choi, Harry L Malech |
Journal | Methods in molecular biology (Clifton, N.J.)
(Methods Mol Biol)
Vol. 1982
Pg. 623-665
( 2019)
ISSN: 1940-6029 [Electronic] United States |
PMID | 31172498
(Publication Type: Journal Article, Research Support, N.I.H., Intramural)
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Chemical References |
- RNA, Guide, CRISPR-Cas Systems
- Reactive Oxygen Species
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Topics |
- CRISPR-Cas Systems
- Cell Differentiation
(genetics, immunology)
- Cell Line
- Cells, Cultured
- Cloning, Molecular
- Gene Editing
(methods)
- Gene Order
- Gene Targeting
- Genetic Vectors
- Granulomatous Disease, Chronic
(genetics, metabolism)
- Hematopoietic Stem Cells
(cytology, metabolism)
- Humans
- Induced Pluripotent Stem Cells
(cytology, metabolism)
- Neutrophils
(cytology, immunology, metabolism)
- RNA, Guide, CRISPR-Cas Systems
- Reactive Oxygen Species
(metabolism)
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