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Journal of Virology, December 2006, p. 12236-12247, Vol. 80, No. 24
0022-538X/06/$08.00+0     doi:10.1128/JVI.01205-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Effect of Adenovirus-Mediated RNA Interference on Endogenous MicroRNAs in a Mouse Model of Multidrug Resistance Protein 2 Gene Silencing{triangledown}

Iñigo Narvaiza ,{dagger},{ddagger} Oscar Aparicio,{dagger} María Vera,§ Nerea Razquin, Sergia Bortolanza, Jesús Prieto, and Puri Fortes*

Division of Gene Therapy and Hepatology, CIMA, University of Navarra, Pio XII 55, 31008 Pamplona, Spain

Received 9 June 2006/ Accepted 19 September 2006

RNA interference with viral vectors that express short hairpin RNAs (shRNAs) has emerged as a powerful tool for functional genomics and therapeutic purposes. However, little is known about shRNA in vivo processing, accumulation, functional kinetics, and side effects related to shRNA saturation of the cellular gene silencing machinery. Therefore, we constructed first-generation recombinant adenoviruses encoding different shRNAs against murine ATP-binding cassette multidrug resistance protein 2 (Abcc2), which is involved in liver transport of bilirubin to bile, and analyzed Abcc2 silencing kinetics. C57/BL6 mice injected with these viruses showed significant impairment of Abcc2 function for up to 3 weeks, as reflected by increased serum bilirubin levels. The lack of Abcc2 function correlated with a specific reduction of Abcc2 mRNA and with high levels of processed shRNAs targeting Abcc2. Inhibition was lost at longer times postinfection, correlating with a decrease in the accumulation of processed shRNAs. This finding suggests that a minimal amount of processed shRNAs is required for efficient silencing in vivo. This system was also used to evaluate the effect of shRNA expression on the saturation of silencing factors. Saturation of the cellular silencing processing machinery alters the accumulation and functionality of endogenous microRNAs (miRNAs) and pre-miRNAs. However, expression of functional exogenous shRNAs did not change the levels of endogenous miRNAs or their precursors. In summary, this work shows that adenoviral vectors can deliver sufficient shRNAs to mediate inhibition of gene expression without saturating the silencing machinery.

* Corresponding author. Mailing address: Division of Gene Therapy and Hepatology, CIMA, University of Navarra, Pio XII 55, 31008 Pamplona, Spain. Phone: 34-948-194700, ext. 4025. Fax: 34-948-194717. E-mail: pfortes{at}unav.es.

{triangledown} Published ahead of print on 4 October 2006.

{dagger} I.N. and O.A. contributed equally to this work.

{ddagger} Present address: Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, Calif.

§ Present address: Department of Medicine, Mount Sinai School of Medicine, New York, N.Y.

Journal of Virology, December 2006, p. 12236-12247, Vol. 80, No. 24
0022-538X/06/$08.00+0     doi:10.1128/JVI.01205-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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  • Abad, X., Vera, M., Jung, S. P., Oswald, E., Romero, I., Amin, V., Fortes, P., Gunderson, S. I. (2008). Requirements for gene silencing mediated by U1 snRNA binding to a target sequence. Nucleic Acids Res 36: 2338-2352 [Abstract] [Full Text]  
  • Witting, S. R., Brown, M., Saxena, R., Nabinger, S., Morral, N. (2008). Helper-dependent Adenovirus-mediated Short Hairpin RNA Expression in the Liver Activates the Interferon Response. J. Biol. Chem. 283: 2120-2128 [Abstract] [Full Text]  


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