Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1939334 | Biochemical and Biophysical Research Communications | 2006 | 7 Pages |
Abstract
Small interfering RNA (siRNA) expression vectors using a Pol III promoter fall into two categories, vectors with a single Pol III promoter that express small hairpin RNA (shRNA) and vectors with two head-to-head (convergent) Pol III promoters that express siRNA. There are technical difficulties in preparing convergent siRNA vectors from cDNA. Here, we report construction of a novel convergent siRNA expression vector, pTHUB. Two XcmI sites were inserted between opposing Pol III promoters. After linearization with XcmI, pTHUB has a single 3â² A overhang at each end that allows direct cloning of partially DNase I digested cDNA fragments (20-30Â bp) tailed with ddT. A derivative method for generating 19Â bp siRNA in pTHUB is also described. The suppression efficiency of the pTHUB vector is comparable to those of conventional shRNA vectors. We have made a siRNA library from a single cDNA. The same approach can be used to construct whole-genome siRNA libraries from cellular cDNA.
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Authors
Cheng Du, Baosheng Ge, Xiaoyan Feng, Wing C. Chan, Timothy W. McKeithan,