Small interfering RNA (siRNA) expression vectors using a Pot III promoter fall into two categories, vectors with a single Pot III promoter that express small hairpin RNA (shRNA) and vectors with two head-to-head (convergent) Pot 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 Pot 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. (c) 2006 Elsevier Inc. All rights reserved.