Multi-gene engineering: simultaneous expression and knockdown of six genes off a single platform

Biotechnol Bioeng. 2007 Apr 1;96(5):821-34. doi: 10.1002/bit.21303.


Increases in our understanding of gene function have greatly expanded the repertoire of possible genetic interventions at our disposal with the consequence that many genetic engineering applications require multiple manipulations in which target genes can be both overexpressed and silenced in a simple and co-ordinated manner. Using synthetic introns as a source of encoding short-interfering RNA (siRNA), we demonstrate that it is possible to simultaneously express both a transgene and siRNA from a single polymerase (Pol) II promoter. By encoding siRNA as an intron between two protein domains requiring successful splicing for functionality, it was possible to demonstrate that splicing was occurring, that the coding genes (exonic transgenes) resulted in functional protein, and that the spliced siRNA-containing lariat was capable of modulating expression of a separate target gene. We subsequently extended this concept to develop pTRIDENT-based multi-cistronic vectors that were capable of co-ordinated expression of up to three siRNAs and three transgenes off a single genetic platform. Such multi-gene engineering technology, enabling concomitant transgene overexpression and target gene knockdown, should be useful for therapeutic, biopharmaceutical production, and basic research applications.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Expression / physiology*
  • Gene Silencing / physiology
  • Genetic Engineering / methods*
  • RNA, Small Interfering / biosynthesis
  • RNA, Small Interfering / physiology*
  • Transgenes / physiology*


  • RNA, Small Interfering