title: Delivery of Genome Editing Reagents to Hematopoietic Stem/Progenitor Cells.
abstract: This unit describes the protocol for the delivery of reagents for targeted genome editing to CD34(+) hematopoietic stem/progenitor cells (HSPCs). Specifically, this unit focuses on the process of thawing and pre-stimulating CD34(+) HSPCs, as well as the details of their electroporation with in vitro-transcribed mRNA-encoding site-specific nucleases [in this case zinc-finger nucleases (ZFNs)]. In addition, discussed is delivery of a gene editing donor template in the form of an oligonucleotide or integrase-defective lentiviral vector (IDLV). Finally, an analysis of cell survival following treatment and downstream culture conditions are presented. While optimization steps might be needed for each specific application with respect to nuclease and donor template amount, adherence to this protocol will serve as an excellent starting point for this further work.
authors: Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California.
authors: Megan D
authors: MD
authors: Hoban
authors: Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California.
authors: Zulema
authors: Z
authors: Romero
authors: Sangamo BioSciences, Richmond, California.
authors: Gregory J
authors: GJ
authors: Cost
authors: Sangamo BioSciences, Richmond, California.
authors: Matthew
authors: M
authors: Mendel
authors: Sangamo BioSciences, Richmond, California.
authors: Michael
authors: M
authors: Holmes
authors: Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California.|Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, Los Angeles, Los Angeles, California.
authors: Donald B
authors: DB
authors: Kohn
mesh_terms: D000818:Animals; D018952:Antigens, CD34; D016503:Drug Delivery Systems / Q000379:methods*; D018274:Electroporation / Q000379:methods*; D015894:Genome, Human; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D016086:Lentivirus; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D018952:Antigens, CD34
keywords: electroporation; genome editing; hematopoietic stem cells; targeted nucleases