用于优化人类造血干细胞和祖细胞CRISPR-Cas9基因编辑过程中体外激活培养条件的方案。
Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells.
作者信息
Della Volpe Lucrezia, Vacca Roberta, Di Micco Raffaella
机构信息
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy.
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy; Vita-Salute San Raffaele University, 20132 Milan, Italy.
出版信息
STAR Protoc. 2025 Mar 31;6(2):103722. doi: 10.1016/j.xpro.2025.103722.
Long-range correction strategies require ex vivo activation of hematopoietic stem and progenitor cells (HSPCs) to engage the homology-directed repair (HDR) mechanism, but prolonged culture causes harmful cellular responses, reducing the long-term functionality of gene-edited (GE) HSPCs. Here, we present a protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human HSPCs. We describe steps for HSPC thawing, ex vivo treatments, gene editing, and downstream in vitro and in vivo analyses to assess the functionality of GE-HSPCs. For complete details on the use and execution of this protocol, please refer to della Volpe et al..
长期校正策略需要对造血干细胞和祖细胞（HSPCs）进行体外激活，以启动同源定向修复（HDR）机制，但长时间培养会引发有害的细胞反应，降低基因编辑（GE）HSPCs的长期功能。在此，我们提出了一种在人类HSPCs的CRISPR-Cas9基因编辑过程中优化体外激活培养条件的方案。我们描述了HSPCs解冻、体外处理、基因编辑以及下游体外和体内分析的步骤，以评估GE-HSPCs的功能。有关本方案使用和执行的完整详细信息，请参考della Volpe等人的研究。
Zotero 插件