Simple and efficient methods for enrichment and isolation of endonuclease modified cells.

The advent of Transcription Activator-Like Effector Nucleases (TALENs), and similar technologies such as CRISPR, provide a straightforward and cost effective option for targeted gene knockout (KO). Yet, there is still a need for methods that allow for enrichment and isolation of modified cells for g...

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Autores principales:	Branden S Moriarity, Eric P Rahrmann, Dominic A Beckmann, Caitlin B Conboy, Adrienne L Watson, Daniel F Carlson, Erik R Olson, Kendra A Hyland, Scott C Fahrenkrug, R Scott McIvor, David A Largaespada
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a67c56a202d14176b28d0ed4ffb24785
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spelling	oai:doaj.org-article:a67c56a202d14176b28d0ed4ffb247852021-11-18T08:20:45ZSimple and efficient methods for enrichment and isolation of endonuclease modified cells.1932-620310.1371/journal.pone.0096114https://doaj.org/article/a67c56a202d14176b28d0ed4ffb247852014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24798371/?tool=EBIhttps://doaj.org/toc/1932-6203The advent of Transcription Activator-Like Effector Nucleases (TALENs), and similar technologies such as CRISPR, provide a straightforward and cost effective option for targeted gene knockout (KO). Yet, there is still a need for methods that allow for enrichment and isolation of modified cells for genetic studies and therapeutics based on gene modified human cells. We have developed and validated two methods for simple enrichment and isolation of single or multiplex gene KO's in transformed, immortalized, and human progenitor cells. These methods rely on selection of a phenotypic change such as resistance to a particular drug or ability to grow in a selective environment. The first method, termed co-transposition, utilizes integration of a piggyBac transposon vector encoding a drug resistance gene. The second method, termed co-targeting, utilizes TALENs to KO any gene that when lost induces a selectable phenotype. Using these methods we also show removal of entire genes and demonstrate that TALENs function in human CD34+ progenitor cells. Further, co-transposition can be used to generate conditional KO cell lines utilizing an inducible cDNA rescue transposon vector. These methods allow for robust enrichment and isolation of KO cells in a rapid and efficient manner.Branden S MoriarityEric P RahrmannDominic A BeckmannCaitlin B ConboyAdrienne L WatsonDaniel F CarlsonErik R OlsonKendra A HylandScott C FahrenkrugR Scott McIvorDavid A LargaespadaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 5, p e96114 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Branden S Moriarity Eric P Rahrmann Dominic A Beckmann Caitlin B Conboy Adrienne L Watson Daniel F Carlson Erik R Olson Kendra A Hyland Scott C Fahrenkrug R Scott McIvor David A Largaespada Simple and efficient methods for enrichment and isolation of endonuclease modified cells.
description	The advent of Transcription Activator-Like Effector Nucleases (TALENs), and similar technologies such as CRISPR, provide a straightforward and cost effective option for targeted gene knockout (KO). Yet, there is still a need for methods that allow for enrichment and isolation of modified cells for genetic studies and therapeutics based on gene modified human cells. We have developed and validated two methods for simple enrichment and isolation of single or multiplex gene KO's in transformed, immortalized, and human progenitor cells. These methods rely on selection of a phenotypic change such as resistance to a particular drug or ability to grow in a selective environment. The first method, termed co-transposition, utilizes integration of a piggyBac transposon vector encoding a drug resistance gene. The second method, termed co-targeting, utilizes TALENs to KO any gene that when lost induces a selectable phenotype. Using these methods we also show removal of entire genes and demonstrate that TALENs function in human CD34+ progenitor cells. Further, co-transposition can be used to generate conditional KO cell lines utilizing an inducible cDNA rescue transposon vector. These methods allow for robust enrichment and isolation of KO cells in a rapid and efficient manner.
format	article
author	Branden S Moriarity Eric P Rahrmann Dominic A Beckmann Caitlin B Conboy Adrienne L Watson Daniel F Carlson Erik R Olson Kendra A Hyland Scott C Fahrenkrug R Scott McIvor David A Largaespada
author_facet	Branden S Moriarity Eric P Rahrmann Dominic A Beckmann Caitlin B Conboy Adrienne L Watson Daniel F Carlson Erik R Olson Kendra A Hyland Scott C Fahrenkrug R Scott McIvor David A Largaespada
author_sort	Branden S Moriarity
title	Simple and efficient methods for enrichment and isolation of endonuclease modified cells.
title_short	Simple and efficient methods for enrichment and isolation of endonuclease modified cells.
title_full	Simple and efficient methods for enrichment and isolation of endonuclease modified cells.
title_fullStr	Simple and efficient methods for enrichment and isolation of endonuclease modified cells.
title_full_unstemmed	Simple and efficient methods for enrichment and isolation of endonuclease modified cells.
title_sort	simple and efficient methods for enrichment and isolation of endonuclease modified cells.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/a67c56a202d14176b28d0ed4ffb24785
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Simple and efficient methods for enrichment and isolation of endonuclease modified cells.

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