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Gene Editing in Chronic Granulomatous Disease.
- Source :
-
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2019; Vol. 1982, pp. 623-665. - Publication Year :
- 2019
-
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.
- Subjects :
- CRISPR-Cas Systems
Cell Differentiation genetics
Cell Differentiation immunology
Cell Line
Cells, Cultured
Cloning, Molecular
Gene Order
Gene Targeting
Genetic Vectors
Granulomatous Disease, Chronic metabolism
Hematopoietic Stem Cells cytology
Hematopoietic Stem Cells metabolism
Humans
Induced Pluripotent Stem Cells cytology
Induced Pluripotent Stem Cells metabolism
Neutrophils cytology
Neutrophils immunology
Neutrophils metabolism
RNA, Guide, CRISPR-Cas Systems
Reactive Oxygen Species metabolism
Gene Editing methods
Granulomatous Disease, Chronic genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1940-6029
- Volume :
- 1982
- Database :
- MEDLINE
- Journal :
- Methods in molecular biology (Clifton, N.J.)
- Publication Type :
- Academic Journal
- Accession number :
- 31172498
- Full Text :
- https://doi.org/10.1007/978-1-4939-9424-3_36