title: Generation of variable and fixed length siRNA from a novel siRNA expression vector.
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 3' A overhang at each end that allows direct cloning of partially DNase I digested cDNA fragments (20-30 bp) tailed with ddT. A derivative method for generating 19 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.
authors: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA. cdu@unmc.edu
authors: Cheng
authors: C
authors: Du
authors: Baosheng
authors: B
authors: Ge
authors: Xiaoyan
authors: X
authors: Feng
authors: Wing C
authors: WC
authors: Chan
authors: Timothy W
authors: TW
authors: McKeithan