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
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author Shiroshita, Kohei
Kobayashi, Hiroshi
Takubo, Keiyo
author_facet Shiroshita, Kohei
Kobayashi, Hiroshi
Takubo, Keiyo
author_sort Shiroshita, Kohei
collection PubMed
description 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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spelling pubmed-102762762023-06-18 Evaluating the function of murine quiescent hematopoietic stem cells following non-homologous end joining-based genome editing Shiroshita, Kohei Kobayashi, Hiroshi Takubo, Keiyo STAR Protoc Protocol 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) Elsevier 2023-06-09 /pmc/articles/PMC10276276/ /pubmed/37300828 http://dx.doi.org/10.1016/j.xpro.2023.102347 Text en © 2023 The Authors. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Shiroshita, Kohei
Kobayashi, Hiroshi
Takubo, Keiyo
Evaluating the function of murine quiescent hematopoietic stem cells following non-homologous end joining-based genome editing
title Evaluating the function of murine quiescent hematopoietic stem cells following non-homologous end joining-based genome editing
title_full Evaluating the function of murine quiescent hematopoietic stem cells following non-homologous end joining-based genome editing
title_fullStr Evaluating the function of murine quiescent hematopoietic stem cells following non-homologous end joining-based genome editing
title_full_unstemmed Evaluating the function of murine quiescent hematopoietic stem cells following non-homologous end joining-based genome editing
title_short Evaluating the function of murine quiescent hematopoietic stem cells following non-homologous end joining-based genome editing
title_sort evaluating the function of murine quiescent hematopoietic stem cells following non-homologous end joining-based genome editing
topic Protocol
url 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
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