Modification of the Creator recombination system for proteomics applications--improved expression by addition of splice sites

BMC Biotechnology
Karen ColwillGregg B Morin

Abstract

Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing ...Continue Reading

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Citations

Mar 10, 2011·Molecular Biotechnology·Natasa SkokoEmanuele Buratti
May 17, 2011·Nature Methods·Karen ColwillSusanne Gräslund
Jul 30, 2011·Nature Methods·Marina OlhovskyKaren Colwill
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Methods Mentioned

BETA
transfection
transfect
PCR
electrophoresis

Software Mentioned

Univector
Creator Splice Creator Splice
Kazusa
mFold
Lumier
Creator
Creator Splice
Nedo
Fluor
BDGP

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