title: Modified gateway system for double shRNA expression and Cre/lox based gene expression.
abstract: BACKGROUND
  The growing need for functional studies of genes has set the stage for the development of versatile tools for genetic manipulations.
  
  
  RESULTS
  Aiming to provide tools for high throughput analysis of gene functions, we have developed a modified short hairpin RNA (shRNA) and gene expression system based on Gateway Technology. The system contains a series of entry and destination vectors that enables easy transfer of shRNA or cDNA into lentiviral expression systems with a variety of selection or marker genes (i.e. puromycin, hygromycin, green fluorescent protein-EGFP, yellow fluorescent protein-YFP and red fluorescent protein-dsRed2). Our shRNA entry vector pENTR.hU6.hH1 containing two tandem human shRNA expression promoters, H1 and U6, was capable of co-expressing two shRNA sequences simultaneously. The entry vector for gene overexpression, pENTR.CMV.ON was constructed to contain CMV promoter with a multiple cloning site flanked by loxP sites allowing for subsequent Cre/lox recombination. Both shRNA and cDNA expression vectors also contained attL sites necessary for recombination with attR sites in our destination expression vectors. As proof of principle we demonstrate the functionality and efficiency of this system by testing expression of several cDNA and shRNA sequences in a number of cell lines.
  
  
  CONCLUSIONS
  Our system is a valuable addition to already existing library of Gateway based vectors and can be an essential tool for many aspects of gene functional studies.
authors.affiliation: University Health Network, Ontario Cancer Institute/Princess Margaret Hospital Site, Toronto, Ontario, Canada.
authors.forename: Nikolina
authors.initials: N
authors.lastname: Radulovich
authors.forename: Lisa
authors.initials: L
authors.lastname: Leung
authors.forename: Ming-Sound
authors.initials: MS
authors.lastname: Tsao
mesh_terms: D002460:Cell Line; D015870:Gene Expression; D018390:Gene Targeting / Q000295:instrumentation / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic
chemical_list: D034741:RNA, Small Interfering; C045073:Cre recombinase; D019426:Integrases