title: Construction of permanently inducible miRNA-based expression vectors using site-specific recombinases.
abstract: BACKGROUND
  RNA interference (RNAi) is a conserved gene silencing mechanism mediated by small inhibitory microRNAs (miRNAs).Promoter-driven miRNA expression vectors have emerged as important tools for delivering natural or artificially designed miRNAs to eukaryotic cells and organisms. Such systems can be used to query the normal or pathogenic functions of natural miRNAs or messenger RNAs, or to therapeutically silence disease genes.
  
  
  RESULTS
  As with any molecular cloning procedure, building miRNA-based expression constructs requires a time investment and some molecular biology skills. To improve efficiency and accelerate the construction process, we developed a method to rapidly generate miRNA expression vectors using recombinases instead of more traditional cut-and-paste molecular cloning techniques. In addition to streamlining the construction process, our cloning strategy provides vectors with added versatility. In our system, miRNAs can be constitutively expressed from the U6 promoter, or inducibly expressed by Cre recombinase. We also engineered a built-in mechanism to destroy the vector with Flp recombinase, if desired. Finally, to further simplify the construction process, we developed a software package that automates the prediction and design of optimal miRNA sequences using our system.
  
  
  CONCLUSIONS
  We designed and tested a modular system to rapidly clone miRNA expression cassettes. Our strategy reduces the hands-on time required to successfully generate effective constructs, and can be implemented in labs with minimal molecular cloning expertise. This versatile system provides options that permit constitutive or inducible miRNA expression, depending upon the needs of the end user. As such, it has utility for basic or translational applications.
authors.affiliation: Center for Gene Therapy, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, 43205, USA.
authors.forename: Sara E
authors.initials: SE
authors.lastname: Garwick-Coppens
authors.forename: Adam
authors.initials: A
authors.lastname: Herman
authors.forename: Scott Q
authors.initials: SQ
authors.lastname: Harper
mesh_terms: D015152:Blotting, Northern; D003001:Cloning, Molecular / Q000379:methods*; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic / Q000235:genetics; D012984:Software
chemical_list: D017931:DNA Primers; D035683:MicroRNAs; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; C077570:Site-specific recombinase
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