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Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming

BACKGROUND: Non-integrating episomal vectors have become an important tool for induced pluripotent stem cell reprogramming. The episomal vectors carrying the “Yamanaka reprogramming factors” (Oct4, Klf, Sox2, and L-Myc + Lin28) are critical tools for non-integrating reprogramming of cells to a pluri...

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Autores principales: Schmitt, Christopher E., Morales, Blanca M., Schmitz, Ellen M. H., Hawkins, John S., Lizama, Carlos O., Zape, Joan P., Hsiao, Edward C., Zovein, Ann C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460403/
https://www.ncbi.nlm.nih.gov/pubmed/28583172
http://dx.doi.org/10.1186/s13287-017-0581-7
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author Schmitt, Christopher E.
Morales, Blanca M.
Schmitz, Ellen M. H.
Hawkins, John S.
Lizama, Carlos O.
Zape, Joan P.
Hsiao, Edward C.
Zovein, Ann C.
author_facet Schmitt, Christopher E.
Morales, Blanca M.
Schmitz, Ellen M. H.
Hawkins, John S.
Lizama, Carlos O.
Zape, Joan P.
Hsiao, Edward C.
Zovein, Ann C.
author_sort Schmitt, Christopher E.
collection PubMed
description BACKGROUND: Non-integrating episomal vectors have become an important tool for induced pluripotent stem cell reprogramming. The episomal vectors carrying the “Yamanaka reprogramming factors” (Oct4, Klf, Sox2, and L-Myc + Lin28) are critical tools for non-integrating reprogramming of cells to a pluripotent state. However, the reprogramming process remains highly stochastic, and is hampered by an inability to easily identify clones that carry the episomal vectors. METHODS: We modified the original set of vectors to express spectrally separable fluorescent proteins to allow for enrichment of transfected cells. The vectors were then tested against the standard original vectors for reprogramming efficiency and for the ability to enrich for stoichiometric ratios of factors. RESULTS: The reengineered vectors allow for cell sorting based on reprogramming factor expression. We show that these vectors can assist in tracking episomal expression in individual cells and can select the reprogramming factor dosage. CONCLUSIONS: Together, these modified vectors are a useful tool for understanding the reprogramming process and improving induced pluripotent stem cell isolation efficiency. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13287-017-0581-7) contains supplementary material, which is available to authorized users.
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spelling pubmed-54604032017-06-07 Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming Schmitt, Christopher E. Morales, Blanca M. Schmitz, Ellen M. H. Hawkins, John S. Lizama, Carlos O. Zape, Joan P. Hsiao, Edward C. Zovein, Ann C. Stem Cell Res Ther Method BACKGROUND: Non-integrating episomal vectors have become an important tool for induced pluripotent stem cell reprogramming. The episomal vectors carrying the “Yamanaka reprogramming factors” (Oct4, Klf, Sox2, and L-Myc + Lin28) are critical tools for non-integrating reprogramming of cells to a pluripotent state. However, the reprogramming process remains highly stochastic, and is hampered by an inability to easily identify clones that carry the episomal vectors. METHODS: We modified the original set of vectors to express spectrally separable fluorescent proteins to allow for enrichment of transfected cells. The vectors were then tested against the standard original vectors for reprogramming efficiency and for the ability to enrich for stoichiometric ratios of factors. RESULTS: The reengineered vectors allow for cell sorting based on reprogramming factor expression. We show that these vectors can assist in tracking episomal expression in individual cells and can select the reprogramming factor dosage. CONCLUSIONS: Together, these modified vectors are a useful tool for understanding the reprogramming process and improving induced pluripotent stem cell isolation efficiency. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13287-017-0581-7) contains supplementary material, which is available to authorized users. BioMed Central 2017-06-05 /pmc/articles/PMC5460403/ /pubmed/28583172 http://dx.doi.org/10.1186/s13287-017-0581-7 Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Method
Schmitt, Christopher E.
Morales, Blanca M.
Schmitz, Ellen M. H.
Hawkins, John S.
Lizama, Carlos O.
Zape, Joan P.
Hsiao, Edward C.
Zovein, Ann C.
Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming
title Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming
title_full Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming
title_fullStr Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming
title_full_unstemmed Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming
title_short Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming
title_sort fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460403/
https://www.ncbi.nlm.nih.gov/pubmed/28583172
http://dx.doi.org/10.1186/s13287-017-0581-7
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