A rapid FACS-based strategy to isolate human gene knockin and knockout clones.

Gene targeting protocols for mammalian cells remain inefficient and labor intensive. Here we describe FASTarget, a rapid, fluorescent cell sorting based strategy to isolate rare gene targeting events in human somatic cells. A fluorescent protein is used as a means for direct selection of targeted cl...

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Autores principales:	João F Mata, Telma Lopes, Rui Gardner, Lars E T Jansen
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e011229d1c35449bad834876954edec2
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spelling	oai:doaj.org-article:e011229d1c35449bad834876954edec22021-11-18T07:26:23ZA rapid FACS-based strategy to isolate human gene knockin and knockout clones.1932-620310.1371/journal.pone.0032646https://doaj.org/article/e011229d1c35449bad834876954edec22012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22393430/?tool=EBIhttps://doaj.org/toc/1932-6203Gene targeting protocols for mammalian cells remain inefficient and labor intensive. Here we describe FASTarget, a rapid, fluorescent cell sorting based strategy to isolate rare gene targeting events in human somatic cells. A fluorescent protein is used as a means for direct selection of targeted clones obviating the need for selection and outgrowth of drug resistant clones. Importantly, the use of a promoter-less, ATG-less construct greatly facilitates the recovery of correctly targeted cells. Using this method we report successful gene targeting in up to 94% of recovered human somatic cell clones. We create functional EYFP-tagged knockin clones in both transformed and non-transformed human somatic cell lines providing a valuable tool for mammalian cell biology. We further demonstrate the use of this technology to create gene knockouts. Using this generally applicable strategy we can recover gene targeted clones within approximately one month from DNA construct delivery to obtaining targeted monoclonal cell lines.João F MataTelma LopesRui GardnerLars E T JansenPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 2, p e32646 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q João F Mata Telma Lopes Rui Gardner Lars E T Jansen A rapid FACS-based strategy to isolate human gene knockin and knockout clones.
description	Gene targeting protocols for mammalian cells remain inefficient and labor intensive. Here we describe FASTarget, a rapid, fluorescent cell sorting based strategy to isolate rare gene targeting events in human somatic cells. A fluorescent protein is used as a means for direct selection of targeted clones obviating the need for selection and outgrowth of drug resistant clones. Importantly, the use of a promoter-less, ATG-less construct greatly facilitates the recovery of correctly targeted cells. Using this method we report successful gene targeting in up to 94% of recovered human somatic cell clones. We create functional EYFP-tagged knockin clones in both transformed and non-transformed human somatic cell lines providing a valuable tool for mammalian cell biology. We further demonstrate the use of this technology to create gene knockouts. Using this generally applicable strategy we can recover gene targeted clones within approximately one month from DNA construct delivery to obtaining targeted monoclonal cell lines.
format	article
author	João F Mata Telma Lopes Rui Gardner Lars E T Jansen
author_facet	João F Mata Telma Lopes Rui Gardner Lars E T Jansen
author_sort	João F Mata
title	A rapid FACS-based strategy to isolate human gene knockin and knockout clones.
title_short	A rapid FACS-based strategy to isolate human gene knockin and knockout clones.
title_full	A rapid FACS-based strategy to isolate human gene knockin and knockout clones.
title_fullStr	A rapid FACS-based strategy to isolate human gene knockin and knockout clones.
title_full_unstemmed	A rapid FACS-based strategy to isolate human gene knockin and knockout clones.
title_sort	rapid facs-based strategy to isolate human gene knockin and knockout clones.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/e011229d1c35449bad834876954edec2
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A rapid FACS-based strategy to isolate human gene knockin and knockout clones.

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