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More siRNA Vectors for RNA Interference

y indications suggest that a great deal of latitude is available in the design of the loop; here we provide our default loop sequence that we find works well.


This vector was developed by Sui and colleagues at Harvard Medical School and has been successfully used to knock down expression of cdk-2 and lamin A/C in HeLa, H1299, U-2 OS and C-33A (cdk-2 only) cells (Sui et al., 2002). Ambion's latest pSilencer siRNA expression vectors, pSilencer 2.0-U6 and pSilencer 3.0-H1, feature two different RNA Polymerase III promoters (Figures 1B and 1C). pSilencer 2.0-U6 contains the human U6 promoter that has been used extensively to express siRNAs and ribozymes in mammalian cells. pSilencer 3.0-H1 features the H1 RNA promoter (a component of RNase P). The pSilencer 2.0-U6 and 3.0-H1 siRNA expression vectors are linearized with two different restriction enzymes to facilitate directional cloning. The linearized vectors are purified to eliminate religation of the vector with the digested fragment. Complementary oligonucleotides encoding siRNA sequences can be readily ligated into the vectors and used to transform E. coli to generate plasmids for siRNA studies (see "Selecting siRNA Sequences to Incorporate into the pSilencer Vectors"). The overhangs are the same for both the pSilencer 2.0-U6 and 3.0-H1 vectors (but are distinct from the linearized pSilencer 1.0-U6 vector); making it possible to subclone the same siRNA insert into either expression vector. This facilitates
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