Generation of a genome scale lentiviral vector library for EF1α promoter-driven expression of human ORFs and identification of human genes affecting viral titer.

The bottleneck in elucidating gene function through high-throughput gain-of-function genome screening is the limited availability of comprehensive libraries for gene overexpression. Lentiviral vectors are the most versatile and widely used vehicles for gene expression in mammalian cells. Lentiviral...

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Autores principales:	Dubravka Škalamera, Mareike Dahmer, Amy S Purdon, Benjamin M Wilson, Max V Ranall, Antje Blumenthal, Brian Gabrielli, Thomas J Gonda
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/d5b5124f4ff94d909044fde07d4f2528
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spelling	oai:doaj.org-article:d5b5124f4ff94d909044fde07d4f25282021-11-18T08:05:16ZGeneration of a genome scale lentiviral vector library for EF1α promoter-driven expression of human ORFs and identification of human genes affecting viral titer.1932-620310.1371/journal.pone.0051733https://doaj.org/article/d5b5124f4ff94d909044fde07d4f25282012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23251614/?tool=EBIhttps://doaj.org/toc/1932-6203The bottleneck in elucidating gene function through high-throughput gain-of-function genome screening is the limited availability of comprehensive libraries for gene overexpression. Lentiviral vectors are the most versatile and widely used vehicles for gene expression in mammalian cells. Lentiviral supernatant libraries for genome screening are commonly generated in the HEK293T cell line, yet very little is known about the effect of introduced sequences on the produced viral titer, which we have shown to be gene dependent. We have generated an arrayed lentiviral vector library for the expression of 17,030 human proteins by using the GATEWAY® cloning system to transfer ORFs from the Mammalian Gene Collection into an EF1alpha promoter-dependent lentiviral expression vector. This promoter was chosen instead of the more potent and widely used CMV promoter, because it is less prone to silencing and provides more stable long term expression. The arrayed lentiviral clones were used to generate viral supernatant by packaging in the HEK293T cell line. The efficiency of transfection and virus production was estimated by measuring the fluorescence of IRES driven GFP, co-expressed with the ORFs. More than 90% of cloned ORFs produced sufficient virus for downstream screening applications. We identified genes which consistently produced very high or very low viral titer. Supernatants from select clones that were either high or low virus producers were tested on a range of cell lines. Some of the low virus producers, including two previously uncharacterized proteins were cytotoxic to HEK293T cells. The library we have constructed presents a powerful resource for high-throughput gain-of-function screening of the human genome and drug-target discovery. Identification of human genes that affect lentivirus production may lead to improved technology for gene expression using lentiviral vectors.Dubravka ŠkalameraMareike DahmerAmy S PurdonBenjamin M WilsonMax V RanallAntje BlumenthalBrian GabrielliThomas J GondaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 12, p e51733 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Dubravka Škalamera Mareike Dahmer Amy S Purdon Benjamin M Wilson Max V Ranall Antje Blumenthal Brian Gabrielli Thomas J Gonda Generation of a genome scale lentiviral vector library for EF1α promoter-driven expression of human ORFs and identification of human genes affecting viral titer.
description	The bottleneck in elucidating gene function through high-throughput gain-of-function genome screening is the limited availability of comprehensive libraries for gene overexpression. Lentiviral vectors are the most versatile and widely used vehicles for gene expression in mammalian cells. Lentiviral supernatant libraries for genome screening are commonly generated in the HEK293T cell line, yet very little is known about the effect of introduced sequences on the produced viral titer, which we have shown to be gene dependent. We have generated an arrayed lentiviral vector library for the expression of 17,030 human proteins by using the GATEWAY® cloning system to transfer ORFs from the Mammalian Gene Collection into an EF1alpha promoter-dependent lentiviral expression vector. This promoter was chosen instead of the more potent and widely used CMV promoter, because it is less prone to silencing and provides more stable long term expression. The arrayed lentiviral clones were used to generate viral supernatant by packaging in the HEK293T cell line. The efficiency of transfection and virus production was estimated by measuring the fluorescence of IRES driven GFP, co-expressed with the ORFs. More than 90% of cloned ORFs produced sufficient virus for downstream screening applications. We identified genes which consistently produced very high or very low viral titer. Supernatants from select clones that were either high or low virus producers were tested on a range of cell lines. Some of the low virus producers, including two previously uncharacterized proteins were cytotoxic to HEK293T cells. The library we have constructed presents a powerful resource for high-throughput gain-of-function screening of the human genome and drug-target discovery. Identification of human genes that affect lentivirus production may lead to improved technology for gene expression using lentiviral vectors.
format	article
author	Dubravka Škalamera Mareike Dahmer Amy S Purdon Benjamin M Wilson Max V Ranall Antje Blumenthal Brian Gabrielli Thomas J Gonda
author_facet	Dubravka Škalamera Mareike Dahmer Amy S Purdon Benjamin M Wilson Max V Ranall Antje Blumenthal Brian Gabrielli Thomas J Gonda
author_sort	Dubravka Škalamera
title	Generation of a genome scale lentiviral vector library for EF1α promoter-driven expression of human ORFs and identification of human genes affecting viral titer.
title_short	Generation of a genome scale lentiviral vector library for EF1α promoter-driven expression of human ORFs and identification of human genes affecting viral titer.
title_full	Generation of a genome scale lentiviral vector library for EF1α promoter-driven expression of human ORFs and identification of human genes affecting viral titer.
title_fullStr	Generation of a genome scale lentiviral vector library for EF1α promoter-driven expression of human ORFs and identification of human genes affecting viral titer.
title_full_unstemmed	Generation of a genome scale lentiviral vector library for EF1α promoter-driven expression of human ORFs and identification of human genes affecting viral titer.
title_sort	generation of a genome scale lentiviral vector library for ef1α promoter-driven expression of human orfs and identification of human genes affecting viral titer.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/d5b5124f4ff94d909044fde07d4f2528
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Generation of a genome scale lentiviral vector library for EF1α promoter-driven expression of human ORFs and identification of human genes affecting viral titer.

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