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DNA Vector-Based shRNA-Expression Systems 231position, whereas the H1 promoter is less strict. The shRNA-expression vectorsare designed particularly that RNA transcripts start with a nucleotide G in thevectors, where it locates within the restriction enzyme ClaI site (see Fig. 2).Specifically selected RNAi-targeting sequences can be easily cloned into anexpression cassette, providing an optimal system for testing endogenous expressionand activity of shRNA. However, one big obstacle for DNA vector-basedRNAi systems is that it takes much time and effort to clone the DNA constructs.To enhance the convenience of constructing a DNA vector-based RNAi systemand facilitate the screening of recombinant clones, all the vectors are furtherimproved by inserting a stuffer of Puro R between the unique cloning sites, ClaIand HindIII, which makes the preparation of the DNA vectors simple and easy byonly removing the stuffer of Puro R DNA fragment with ClaI and HindIII doubledigestion (see Fig. 3) (Note 2) (38).3.1.2. Construction of Improved shRNA-Expression VectorsThe shRNA-expression vectors, including pHsH1, pHsU6, pMmH1, andpMmU6 (see Fig. 2), are constructed by PCR-based cloning method. The RNAPol III-regulated type III promoters, including H1 and U6 from human (Hs)and mouse (Mm), are amplified by standard PCR reaction using syntheticoligonucleotides, which are purchased from local commercial suppliers (seeNote 3). The oligonucleotides used for amplification of the HsH1, HsU6,MmH1, and MmU6 are:HsH1-S: 5′-GGAATTCGAACGCTGACGTCATCAAC-3′ and HsH1-AS:5′-CCATCGATAAAGAGTGGTCTCATACAG-3′; HsU6-S:5′-GGAATTCAAGGTCGGGC AGG AAGAGG-3′ and HsU6-AS:5′-CCCAAGCTTCCATCGATGTTTCGTCCTTTCCACAAGATAT-3′; MmH1-S:5′-GGAATTCCGCTCTTGAAGGACGACGTCATC-3′ and MmH1-AS:5′-CCATCGATAGGGTGTAGACCGGCCGCCAC-3′; MmU6-S:5′-GGAATTCATCCGACGCCGCCATCTCTAGG-3′ and MmU6-AS:5′-CCATCGATCAAGGCTTTTCTCCAAGGGATA-3′.To simplify the construction procedures, the amplification product of HsU6promoter is first treated with EcoRI and HindIII restriction enzymes, thencloned into an EcoRI/HindIII-digested pGEM-7ZF(+) vector (see Note 4), andthe resulting plasmid is designated as pHsU6. Subsequently, the other amplificationproducts including HsH1, MmH1, and MmU6 promoters are treated withEcoRI and ClaI restriction enzymes, subcloned into an EcoRI/ClaI-digestedpHsU6 vector to substitute the HsU6 promoter, and the resulting plasmids arecalled as pHsH1, pMmH1, and pMmU6. To construct the improved cloningvectors, including pHsH1puro, pHsU6puro, pMmH1puro, and pMmU6puro(see Fig. 3), a ClaI/HindIII-treated Puro R DNA fragment isolated from