mRNA transfection of mouse and human neural stem cell cultures.

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the in...

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Autores principales:	Samuel McLenachan, Dan Zhang, Ana Belén Alvarez Palomo, Michael J Edel, Fred K Chen
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/691578f40808496b910b2f0f0c0b4bc2
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spelling	oai:doaj.org-article:691578f40808496b910b2f0f0c0b4bc22021-11-18T08:40:20ZmRNA transfection of mouse and human neural stem cell cultures.1932-620310.1371/journal.pone.0083596https://doaj.org/article/691578f40808496b910b2f0f0c0b4bc22013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24386231/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.Samuel McLenachanDan ZhangAna Belén Alvarez PalomoMichael J EdelFred K ChenPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 12, p e83596 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Samuel McLenachan Dan Zhang Ana Belén Alvarez Palomo Michael J Edel Fred K Chen mRNA transfection of mouse and human neural stem cell cultures.
description	The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.
format	article
author	Samuel McLenachan Dan Zhang Ana Belén Alvarez Palomo Michael J Edel Fred K Chen
author_facet	Samuel McLenachan Dan Zhang Ana Belén Alvarez Palomo Michael J Edel Fred K Chen
author_sort	Samuel McLenachan
title	mRNA transfection of mouse and human neural stem cell cultures.
title_short	mRNA transfection of mouse and human neural stem cell cultures.
title_full	mRNA transfection of mouse and human neural stem cell cultures.
title_fullStr	mRNA transfection of mouse and human neural stem cell cultures.
title_full_unstemmed	mRNA transfection of mouse and human neural stem cell cultures.
title_sort	mrna transfection of mouse and human neural stem cell cultures.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/691578f40808496b910b2f0f0c0b4bc2
work_keys_str_mv	AT samuelmclenachan mrnatransfectionofmouseandhumanneuralstemcellcultures AT danzhang mrnatransfectionofmouseandhumanneuralstemcellcultures AT anabelenalvarezpalomo mrnatransfectionofmouseandhumanneuralstemcellcultures AT michaeljedel mrnatransfectionofmouseandhumanneuralstemcellcultures AT fredkchen mrnatransfectionofmouseandhumanneuralstemcellcultures
_version_	1718421449960587264

mRNA transfection of mouse and human neural stem cell cultures.

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