Lund, Anne Mathilde1; Kildegaard, Helene Faustrup3; Petersen, Maja Borup Kjær8; Rank, Julie5; Hansen, Bjarne Gram1; Andersen, Mikael Rørdam1; Mortensen, Uffe Hasbro1
1 Department of Systems Biology, Technical University of Denmark2 Network Engineering of Eukaryotic Cell Factories, Department of Systems Biology, Technical University of Denmark3 Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark4 CHO Cell Line Engineering and Design, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark5 Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark6 Systems Biotechnology, Department of Systems Biology, Technical University of Denmark7 Eucaryotic Molecular Cell Biology, Department of Systems Biology, Technical University of Denmark8 Technical University of Denmark
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.