Delivery of Genome Editing Reagents to Hematopoietic Stem/Progenitor Cells. (13th February 2018)

• Record Type: Journal Article
• Authors: Hoban, Megan D.; Romero, Zulema; Cost, Gregory J.; Mendel, Matthew; Holmes, Michael; Kohn, Donald B.
• Editors: Bhatia, Mick; Elefanty, Andrew G.; Fisher, Susan J.; Patient, Roger; Schlaeger, Thorsten; Snyder, Evan Y.
• Is Part Of: Current protocols in stem cell biology. Volume 36(2016)
• Page Start: 5B.4.1
• Page End: 5B.4.10
• Publication Date: 2018-02-13
• Subjects: hematopoietic stem cells -- targeted nucleases -- electroporation -- genome editing; Stem cells -- Laboratory manuals; Stem cells -- Physiology; Stem Cells; Stem cells; Laboratory Manual; Laboratory manuals; 571.5
• DOI: 10.1002/9780470151808.sc05b04s36
• Languages: English
• ISSNs: 1941-7322

Abstract

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. © 2016 by John Wiley & Sons, Inc.