Delivery of Genome Editing Reagents to Hematopoietic Stem/Progenitor Cells.
Delivery of Genome Editing Reagents to Hematopoietic Stem/Progenitor Cells.
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DOI：
10.1002/9780470151808.sc05b04s36
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发表时间：
2016-02-03
影响因子：
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通讯作者：
Kohn, Donald B
中科院分区：
文献类型：
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作者：
Hoban, Megan D;Romero, Zulema;Kohn, Donald B
本单位介绍了将用于定向基因组编辑的试剂输送到CD34(+)造血干细胞/祖细胞(HSPC)的方案。具体地说，本单元侧重于解冻和预刺激CD34(+)HPC的过程，以及它们与体外转录的mRNA编码位点特异性核酸酶[在这种情况下是锌指核酸酶(ZFN)]的电穿孔细节。此外，还讨论了以寡核苷酸或整合酶缺陷慢病毒载体(IDLV)的形式递送基因编辑供体模板。最后，对治疗后的细胞存活和下游培养条件进行了分析。虽然每个特定应用可能需要针对核酸酶和供体模板数量的优化步骤，但遵守该协议将为进一步的工作提供一个很好的起点。
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.