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Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells

The main methods for producing genetically engineered cells use viral vectors for which safety issues and manufacturing costs remain a concern. In addition, selection of desired cells typically relies on the use of cytotoxic drugs with long culture times. Here, we introduce an efficient non-viral ap...

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Autores principales: Kacherovsky, Nataly, Harkey, Michael A., Blau, C. Anthony, Giachelli, Cecilia M., Pun, Suzie H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367214/
https://www.ncbi.nlm.nih.gov/pubmed/22402491
http://dx.doi.org/10.1093/nar/gks213
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author Kacherovsky, Nataly
Harkey, Michael A.
Blau, C. Anthony
Giachelli, Cecilia M.
Pun, Suzie H.
author_facet Kacherovsky, Nataly
Harkey, Michael A.
Blau, C. Anthony
Giachelli, Cecilia M.
Pun, Suzie H.
author_sort Kacherovsky, Nataly
collection PubMed
description The main methods for producing genetically engineered cells use viral vectors for which safety issues and manufacturing costs remain a concern. In addition, selection of desired cells typically relies on the use of cytotoxic drugs with long culture times. Here, we introduce an efficient non-viral approach combining the Sleeping Beauty (SB) Transposon System with selective proliferation of engineered cells by chemically induced dimerization (CID) of growth factor receptors. Minicircles carrying a SB transposon cassette containing a reporter transgene and a gene for the F36VFGFR1 fusion protein were delivered to the hematopoietic cell line Ba/F3. Stably-transduced Ba/F3 cell populations with >98% purity were obtained within 1 week using this positive selection strategy. Copy number analysis by quantitative PCR (qPCR) revealed that CID-selected cells contain on average higher copy numbers of transgenes than flow cytometry-selected cells, demonstrating selective advantage for cells with multiple transposon insertions. A diverse population of cells is present both before and after culture in CID media, although site-specific qPCR of transposon junctions show that population diversity is significantly reduced after selection due to preferential expansion of clones with multiple integration events. This non-viral, positive selection approach is an attractive alternative for producing engineered cells.
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spelling pubmed-33672142012-06-05 Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells Kacherovsky, Nataly Harkey, Michael A. Blau, C. Anthony Giachelli, Cecilia M. Pun, Suzie H. Nucleic Acids Res Methods Online The main methods for producing genetically engineered cells use viral vectors for which safety issues and manufacturing costs remain a concern. In addition, selection of desired cells typically relies on the use of cytotoxic drugs with long culture times. Here, we introduce an efficient non-viral approach combining the Sleeping Beauty (SB) Transposon System with selective proliferation of engineered cells by chemically induced dimerization (CID) of growth factor receptors. Minicircles carrying a SB transposon cassette containing a reporter transgene and a gene for the F36VFGFR1 fusion protein were delivered to the hematopoietic cell line Ba/F3. Stably-transduced Ba/F3 cell populations with >98% purity were obtained within 1 week using this positive selection strategy. Copy number analysis by quantitative PCR (qPCR) revealed that CID-selected cells contain on average higher copy numbers of transgenes than flow cytometry-selected cells, demonstrating selective advantage for cells with multiple transposon insertions. A diverse population of cells is present both before and after culture in CID media, although site-specific qPCR of transposon junctions show that population diversity is significantly reduced after selection due to preferential expansion of clones with multiple integration events. This non-viral, positive selection approach is an attractive alternative for producing engineered cells. Oxford University Press 2012-06 2012-02-08 /pmc/articles/PMC3367214/ /pubmed/22402491 http://dx.doi.org/10.1093/nar/gks213 Text en © The Author(s) 2012. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Kacherovsky, Nataly
Harkey, Michael A.
Blau, C. Anthony
Giachelli, Cecilia M.
Pun, Suzie H.
Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells
title Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells
title_full Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells
title_fullStr Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells
title_full_unstemmed Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells
title_short Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells
title_sort combination of sleeping beauty transposition and chemically induced dimerization selection for robust production of engineered cells
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367214/
https://www.ncbi.nlm.nih.gov/pubmed/22402491
http://dx.doi.org/10.1093/nar/gks213
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