Format

Send to

Choose Destination
J Virol Methods. 2004 Dec 1;122(1):73-7.

Increased protein expression from adenoviral shuttle plasmids and vectors by insertion of a small chimeric intron sequence.

Author information

1
Department of Neurology, University of Jena, Erlanger Allee 101, D-07747 Jena, Germany.

Abstract

Adenoviruses are widely used as gene transfer vehicles because they can be produced at high titers, they have a large transgene capacity, and can transduce both dividing and non-dividing cells. One disadvantage of adenoviral vectors is the narrow therapeutic window due to a dose-dependent humoral as well as a T-cell dependent host immune response directed against the transduced cells, that leads to a reduction of transgene expression with time. By increasing the levels of protein expression from transcription units, vector titres may be decreased without a significant loss of transgene expression. Introns are required for efficient expression of many protein coding genes. In addition, splicing signals are required for some genes in order to be translated. Therefore, a chimeric intron sequence was introduced at the 3' end of the transgenes to study its effect on protein expression from adenoviral vector constructs. Transfection of 293 cells with the adenoviral shuttle plasmids pMH4-EGFP-Int, pMH4-E314.7-Int, pMH4-BclX(L)-Int and pMH4-Luc-Int lead to a 1.8-20-fold increase in protein expression as compared to constructs lacking an intron. Injection of Ad-CMV-Luc-Int into the brain of C57Bl/6 mice results in an approximately three-fold increase of luciferase activity as compared to Ad-CMV-Luc. In conclusion, insertion of an intron sequence leads to a significant increase in transgene expression both in vitro and in vivo, thus allowing for reduction of the adenoviral vector dose used.

PMID:
15488623
DOI:
10.1016/j.jviromet.2004.08.005
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center