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230 Cheng and Chang3. Handheld ultraviolet (UV) lamp (VL-4.L, Vilber Lourmat, Marne-la-Vallee, France).4. UV image system (UV illuminator, Vilber Lourmat, Marne-la-Vallee, France).5. Spectrophotometer (Beckman DU 640, Beckman Instruments, Fullerton, CA).6. Microplate reader (Dynatech MR5000, Dynatech Laboratories, Chantilly, VA).7. Luminometer (MiniLumat LB 9506, EG&G Berthold, Wildbach, Germany).8. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis apparatus (MightySmall II 8 × 7 cm 2 , Hoefer Scientific Instruments, San Francisco, CA).9. Electrophoresis power supply (EPS 1000, Amersham Pharmacia Biotech, Uppsala,Sweden).10. Semidry transfer apparatus (Semiphor transphor unit, Amersham Pharmacia Biotech).11. Automated DNA sequencer (ABI PRISM 377 DNA sequencer, Applied Biosystems,Foster, CA).3. MethodsThe methods described in this section outline (1) the structural features andconstruction of improved shRNA-expression vectors, (2) the molecular characteristicsof designed and selected RNAi-targeting sequences, (3) the proceduresfor cloning shRNA-expression vectors, and (4) the approaches for assessinggene silencing efficiency by shRNA-expression vectors.3.1. Structural Features and Construction of ImprovedshRNA-Expression Vectors3.1.1. Structural Features of Improved shRNA-Expression VectorsThe functional active siRNA, either in vivo identified or in vitro synthesized,is a small 21–23-nt RNA duplex with symmetrical 2-nt 3′-overhangs (3,4). Inaddition, the long dsRNAs stimulate a serious cytotoxic response through activationof the dsRNA-dependent protein kinase and 2′,5′-oligoadenylate synthetasein mammalian cells (16–20). However, this nonselective cytotoxic effectcan be overcome by directly applying small dsRNAs with the size smaller than30-nt in length, including synthetic or expressed siRNAs and shRNAs. TheRNA Pol III-regulated type III promoters, especially H1 and U6 from humanand mouse, have been used most frequently, because they transcribe the RNAfrom a defined start site (+1) and terminate at a run of 5–6 Ts. As well as, theycan efficiently express small-RNA transcripts without posttranscriptional modificationincluding 5′-capping and 3′-polyadenylation (34–36). Thus, these promotersare suitable for expression of the defined small-RNA transcripts with thefeatures fulfilling the aforementioned criteria.In addition, to make the construction of shRNA-expression vectors simple andconvenient, all the vectors are constructed to contain the same unique cloningsites, ClaI and HindIII, for cloning the RNAi-targeting sequences (see Note 1).Because U6 promoter transcribes preferentially from a “G” nucleotide at the +1