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228 Cheng and ChangFig. 2. Sequences and structures of RNA Pol III-controlled type III promoter-basedshRNA-expression vectors. The human H1 (HsH1) and U6 (HsU6), and mouse H1(MmH1) and U6 (MmU6) promoters are isolated from human and mouse genomicDNAs by PCR amplification, and cloned into the pGEM-7ZF(+) vector. The resultingDNA constructs are designated as pHsH1 (A), pHsU6 (B), pMmH1 (C), and pMmU6(D) vectors. These four DNA vectors all contain the same unique restriction enzyme ClaIand HindIII sites for cloning the shRNA-coding sequences. In these four DNA vectors,the proximal sequence element is in white and shaded in blue, TATA box is in bold andshaded in green, restriction enzyme sites of EcoRI (GAATTC), ClaI (ATCGAT), andHindIII (AAGCTT) are underlined and in purple and bold, and G is the transcriptioninitiation site (+1).2. 10 mM Deoxynucleoside triphosphate mixtures (Promega) stored at −30°C.3. PCR reagents, including Taq DNA polymerase and 10X reaction buffer withMgCl 2(Promega) stored at −30°C.2.4. Transfection and Functional Assessments1. Lipofectamine 2000 (Invitrogen, Carlsbad, CA) stored at 4°C.2. TRI Reagent (Molecular Research Center, Cincinnati, OH) stored at 4°C.3. Protein lysis buffer: 50 mM NaCl, 50 mM Tris-HCl, 2 mM EDTA, 0.5% sodiumdeoxycholate, 1% NP-40 (Roche Molecular Biochemicals, Mannheim, Germany),and 0.1% SDS, pH 7.4, stored at room temperature.4. Protease inhibitors (Roche) stored in aliquots at −80°C.