Andersen, Mikael Rørdam

Hansen, Bjarne Gram

Holm, Dorte Koefoed

Kildegaard, Helene Faustrup

Lund, Anne Mathilde

Mortensen, Uffe Hasbro

The development in the field of mammalian cell factories require fast and high-throughput methods, this means a high need for simpler and more efficient cloning techniques. For optimization of protein expression by genetic engineering and for allowing metabolic engineering in mammalian cells, a new versatile expression vector system was developed. This vector system applies the ligation-free uracilexcision cloning technique to construct mammalian expression vectors of multiple parts and with maximum flexibility

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Downloaded from orbit.dtu.dk on: Dec 19, 2017A validated system for ligation-free USER™ -based assembly of expression vectors formammalian cell engineeringLund, Anne Mathilde; Kildegaard, Helene Faustrup; Hansen, Bjarne Gram; Holm, Dorte Koefoed;Andersen, Mikael Rørdam; Mortensen, Uffe HasbroPublication date:2013Document VersionPeer reviewed versionLink back to DTU OrbitCitation (APA):Lund, A. M., Kildegaard, H. F., Hansen, B. G., Holm, D. K., Andersen, M. R., & Mortensen, U. H. (2013). Avalidated system for ligation-free USER™ -based assembly of expression vectors for mammalian cellengineering. Poster session presented at 23rd ESACT meeting, Lille, France.IntroductionThe development in the field of mammalian cell factories require fast and high-throughput methods, this means a high need for simpler and more efficient cloning techniques. For optimization of protein expression by genetic engineering and for allowing metabolic engine-ering in mammalian cells, a new versatile expression vector system was developed. This vector system applies the ligation-free uracil-excision cloning technique to construct mammalian expression vectors of multiple parts and with maximum flexibility. Vector assembly platform allows high-throughput generation of genetic constructsReferences1. B.G. Hansen et al., Versatile enzyme expression and characterization system for Aspergillus nidulans, with the Penicillium brevicompactum polyketide synthase gene from the mycophenolic acid gene cluster as a test case, Appl Environ Microbiol. (2011) 77(9):3044-51. 2.  H.H. Nour-Eldin et al., USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories, Methods Mol. Biol. (2010) 643:185-200.3. M. Nørholm, A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering, BMC Biotechnol. (2010) 10(1):21.4. S.S. Tate et al., Secreted alkaline phosphatase: an internal standard for expression of injected mRNAs in the Xenopus oocyte, FASEB J. (1990) 4(2): 227-231. AcknowledgementThanks to the Danish Cancer Society Research Center for providing template plasmids, the U-2 OS and HEK293 cell line. Thanks to Novo Nordisk for kindly providing the CHO-K1 cell line and thanks to the Novo Nordisk Foundation Center for Protein Research for providing the genes of the fluorescent proteins. A special thank to the 2011 iGEM team DTU-Denmark-2. Finally, a thanks to the Core Facilities of Integrated Microscopy at Copenhagen University for use of confocal microscopy.A validated system for ligation-free uracil-excision based assembly of expression vectors for mammalian cell engineeringAnne Mathilde Lund1, *, Helene Faustrup Kildegaard2, Bjarne Gram Hansen3, Dorte Koefoed Holm1, Mikael Rørdam Andersen1, Uffe Hasbro Mortensen1 (1) Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark; (2) Current address: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hoersholm, Denmark; (3) Current address: Novozymes A/S, Bagsvaerd, Denmark(*) amalu@bio.dtu.dkThe assembly method has been tested with >25 generic parts Mergedβ -1.4 GT α-2.6 STGalNAcT1FluorescenceA B CDAPItrans-golgi TGNmedial - golgiCalreticulin/KDELERDc-Ha-RasCOX-VIIImitocondria plasma membranePTS1NLSnucleus peroxisomesE F G HGOIDNA elementsVector backbonesPromoterGOITerminatorA BPromoterGOITerminatorGOIHybridizationE. coli transformantGOIUracil-excisionOne-step transformation+ +P-GOI-T+Promoter - GOI - TerminatorGOIP G TCP TP TTPPromoter - GOI - TerminatorHigh efficiency one step ligation-free uracil-excision cloningTAAGGCTAATTCCGATGCAGCGAACGTCGCTCACGCTAAGTGCGAAGTGCGATCACGCTAATTCCGATAAGGCTAATTAAGCTAATTCGATerminatorPromoter  GOIPre-prepared PCR DNA fragmentsUSERTM enzymePromoterACGTCGCUTGCAGCGA AGTGCGATUCACGCTAGOIAGTGCGAUTCACGCTA ATTCCGATUAAGGCTABackboneATTAAGCUTAATTCGA ACGTCGCTUGCAGCGABackboneTerminatorATTCCGAUTAAGGCTA ATTAAGCTUAATTCGATAATTCGAATTAAGCACGTCGCTGCAGCGAProof-of-concept: Successful protein targeting to organelles PerspectivesThe developed vector assembly system already facilitates high-throughput construction of plasmid DNA for  system-wide studies of CHO cell lines and will eliminate bottlenecks within genomic studies of mammalian cells. In the future, the system is planned to include site-specific integration and knockout cassettes.Promoters: • CMV • PGK  • SV40 Terminators: • BGH polyA • SV40 polyA • hGH polyA Markers: • Hygromycin • Neomycin Targeting signals: • Nucleus • Peroxisomes • Mitochondria • Plasma membrane • ER • Medial Golgi • Trans Golgi • TGN Golgi • Secretion signals Reporters: • eGFP • eYFP • eCFP • mCherry • ccdB • SEAP Other elements: • IRES • ccdB Epitope-tags: • c-Myc tag • HA tag • His tag 93 % 96 % 100 % 100 % 7 DNA fragments 6 DNA fragments 5 DNA fragments 1 DNA fragment 101 141 339 1000 5 7 DNA fragments 6 DNA fragments 5 DNA fragments 1 DNA fragment Negative control Promoter - GOI - terminatorExpression cassettes A, B, and C below. Promoter GOI TerminatorPromoter GOI MarkerTerminatorG0AGTGCGAUUGTGTCAGA5’3’ OI 5’3’Uracil-excision primer PCRA library of the most commonly used generic parts for transient and stable plasmid expression in mam-malian cells have been created and tested. All parts are ready-to-clone DNA fragments, which allow fast and versatile vector construction with the possibilities of bi-cistronic expression, compartmentalisation, fusion to reporter gene or tags.Transient expression was validated in the U-2 OS cell line by a set of vectors for localisation to the major compartments. Expression of fluorescent proteins and protein secretion of se-creted alkaline phosphatase4 (SEAP) were acchived in HEK293 cells as well as in CHO-K1 cell lines. Furthermore, constructed vectors have successfully been applied for generating stable CHO-K1 cell lines for protein expression by use of selection pressure with Neomycin.Ready-to-clone DNA fragment libraryA novel cassette free assembly system was created for solely PCR-based assembly of multiple parts. The simplicity and flexibility of the uracil-excision cloning technique facilitated the adaption of the vector system into high-throughput cloning of a single element or versatile assembly of multiple elements. An expression vector of seven generic elements can be assembled and ready for tranfection within three working days. Uracil-excision is a PCR-based cloning method that employs the USERTM enzyme, a mix-ture of a Uracil DNA glycosylase and the DNA glycosylase-lyase Endonuclease VIII. The method generates long complementary overhangs that anneal to each other to form a stable hybridisation product that can be used to transform E. coli without prior ligation1,2. The method has advantages of permitting additional modification in the assembly process including point mutations, insertion, deletion as well as seamless fusion of DNA fragments3. With all vector parts amplified separately with standardised overhangs, they can be combi-ned as preferred in an fast and easy manner.Each vector part is PCR-amplified  with uracil-primers encoding stan-dardised overhangs. Ten different con-structs was assemb-led in each catagory.Vector assembly efficiencyAverage number of E. coli transformansThree transformants tes-ted for each of the ten contructs.

A validated system for ligation-free USER™ -based assembly of expression vectors for mammalian cell engineering

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A validated system for ligation-free USER™ -based assembly of expression vectors for mammalian cell engineering

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