A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.

A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments an...

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Autores principales:	Anne Mathilde Lund, Helene Faustrup Kildegaard, Maja Borup Kjær Petersen, Julie Rank, Bjarne Gram Hansen, Mikael Rørdam Andersen, Uffe Hasbro Mortensen
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/181d730b6705404b876e3f3957db1fb4
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spelling	oai:doaj.org-article:181d730b6705404b876e3f3957db1fb42021-11-18T08:17:43ZA versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.1932-620310.1371/journal.pone.0096693https://doaj.org/article/181d730b6705404b876e3f3957db1fb42014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24879460/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors.Anne Mathilde LundHelene Faustrup KildegaardMaja Borup Kjær PetersenJulie RankBjarne Gram HansenMikael Rørdam AndersenUffe Hasbro MortensenPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 5, p e96693 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Anne Mathilde Lund Helene Faustrup Kildegaard Maja Borup Kjær Petersen Julie Rank Bjarne Gram Hansen Mikael Rørdam Andersen Uffe Hasbro Mortensen A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.
description	A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors.
format	article
author	Anne Mathilde Lund Helene Faustrup Kildegaard Maja Borup Kjær Petersen Julie Rank Bjarne Gram Hansen Mikael Rørdam Andersen Uffe Hasbro Mortensen
author_facet	Anne Mathilde Lund Helene Faustrup Kildegaard Maja Borup Kjær Petersen Julie Rank Bjarne Gram Hansen Mikael Rørdam Andersen Uffe Hasbro Mortensen
author_sort	Anne Mathilde Lund
title	A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.
title_short	A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.
title_full	A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.
title_fullStr	A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.
title_full_unstemmed	A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.
title_sort	versatile system for user cloning-based assembly of expression vectors for mammalian cell engineering.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/181d730b6705404b876e3f3957db1fb4
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A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.

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