A recombinase-based palindrome generator capable of producing randomized shRNA libraries.
Short hairpin RNA (shRNA)-expressing vectors have been shown stably to knock-down directed targets through RNA interference (RNAi). RNAi has emerged as a powerful tool for reverse genetic screens by assaying cellular phenotypes after exposure to libraries of pooled shRNAs directed against all or a subset of cellular mRNAs. Recently, noncoding RNAs have been recognized as major arbiters of cellular phenotypes. The scope and diversity of noncoding RNAs greatly enlarge the target pool for reversible genetic screens and underscore the desirability as screening tools of complex RNAi libraries, such as randomized RNAi libraries. We describe a novel approach to generate randomized shRNAs that takes advantage of a stable plasmid intermediate that arises in a FLP recombinase system. The ability of this system to generate randomized palindromes represents a new technology for shRNA generation including random shRNAs that cannot be produced synthetically. We describe this plasmid system and its use in generating randomized shRNAs from input 20-bp oligomers, and validate the functionality of a shRNA produced using this approach to knock-down estrogen receptor alpha expression.
Nichols
Mark
M
Department of Pharmacology, 2.26 Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA 15213, United States. mnichols@pitt.edu
Steinman
Richard A
RA
D003001:Cloning, Molecular / Q000379:methods; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism; D055785:Gene Knockdown Techniques / Q000379:methods*; D015723:Gene Library; D055029:Inverted Repeat Sequences; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results
D047628:Estrogen Receptor alpha; D034741:RNA, Small Interfering; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase