title: Modification of the Creator recombination system for proteomics applications--improved expression by addition of splice sites.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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 in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased.
  
  
  CONCLUSIONS
  The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics.
authors.affiliation: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. colwill@mshri.on.ca
authors.forename: Karen
authors.initials: K
authors.lastname: Colwill
authors.forename: Clark D
authors.initials: CD
authors.lastname: Wells
authors.forename: Kelly
authors.initials: K
authors.lastname: Elder
authors.forename: Marilyn
authors.initials: M
authors.lastname: Goudreault
authors.forename: Kadija
authors.initials: K
authors.lastname: Hersi
authors.forename: Sarang
authors.initials: S
authors.lastname: Kulkarni
authors.forename: W Rod
authors.initials: WR
authors.lastname: Hardy
authors.forename: Tony
authors.initials: T
authors.lastname: Pawson
authors.forename: Gregg B
authors.initials: GB
authors.lastname: Morin
mesh_terms: D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D019426:Integrases; D016366:Open Reading Frames; D040901:Proteomics / Q000379:methods*; D022821:RNA Splice Sites; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D014764:Viral Proteins
chemical_list: D022821:RNA Splice Sites; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases