Evaluating the function of murine quiescent hematopoietic stem cells following non-homologous end joining-based genome editing

Preculture is indispensable for achieving highly efficient non-homologous end joining (NHEJ)-based genome editing. Here, we present a protocol for optimizing genome editing conditions for murine hematopoietic stem cells (HSCs) and evaluating their function following NHEJ-based genome editing. We des...

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Detalles Bibliográficos
Autores principales: Shiroshita, Kohei, Kobayashi, Hiroshi, Takubo, Keiyo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10276276/
https://www.ncbi.nlm.nih.gov/pubmed/37300828
http://dx.doi.org/10.1016/j.xpro.2023.102347
Descripción
Sumario:Preculture is indispensable for achieving highly efficient non-homologous end joining (NHEJ)-based genome editing. Here, we present a protocol for optimizing genome editing conditions for murine hematopoietic stem cells (HSCs) and evaluating their function following NHEJ-based genome editing. We describe steps for sgRNA preparation, cell sorting, preculture, and electroporation. We then detail post-editing culture and transplanting of bone marrow. This protocol can be used to study genes related to HSC quiescence. For complete details on the use and execution of this protocol, please refer to Shiroshita et al.(1)

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