A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

• DOI: 10.1371/journal.pone.0096693
• Publication Date: 2014-05-30T20:14:10Z
• Authors: Anne Mathilde Lund; Helene Faustrup K...; Maja Borup Kjær P...; Julie Rank; Bjarne Gram Hansen; Mikael Rørdam And...; Uffe Hasbro Morte...
• Keywords: Multiple cloning site; Selectable marker; Cloning (programming); mCherry; Expression cassette; Cloning vector

A new versatile mammalian vector system for protein production, cell biology analyses, and factory engineering was developed. The applies the ligation-free uracil-excision based technique – USER cloning to rapidly construct expression vectors of multiple DNA fragments with maximum flexibility, both choice backbone cargo. includes a set basic toolbox containing multitude building blocks including promoters, terminators, selectable marker- reporter genes, sequences encoding an internal ribosome entry site, cellular localization signals epitope- purification tags. Building in can be easily combined as they contain defined tested Flexible Assembly Sequence Tags, FASTs. FASTs allows rapid swaps gene, promoter or selection marker existing plasmids simple construction proteins, which are fused fluorescence-, purification-, localization-, epitope assembly platform currently up seven single step correct directionality efficiency above 90%. functionality FAST validated by transient fluorescent model proteins CHO, U-2-OS HEK293 lines. In this test, we included many most common elements heterologous gene cells, addition is fully extendable other users. designed facilitate high-throughput genome-scale studies such newly sequenced CHO lines, through ability generate high-fidelity customizable vectors.