Generation of variable and fixed length siRNA from a novel siRNA expression vector

Biochem Biophys Res Commun. 2006 Jun 23;345(1):99-105. doi: 10.1016/j.bbrc.2006.04.061. Epub 2006 Apr 25.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Gene Silencing*
  • Genetic Engineering / methods*
  • Genetic Variation / genetics*
  • Genetic Vectors / genetics*
  • Molecular Sequence Data
  • Promoter Regions, Genetic / genetics*
  • RNA, Small Interfering / genetics*

Substances

  • RNA, Small Interfering