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Nucleic Acids Research Advance Access originally published online on April 10, 2007
Nucleic Acids Research 2007 35(9):e65; doi:10.1093/nar/gkm152
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Nucleic Acids Research, 2007, Vol. 35, No. 9 e65
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

A hybrid CMV-H1 construct improves efficiency of PEI-delivered shRNA in the mouse brain

Zahra Hassani1,{dagger}, Jean-Christophe François2,3,{dagger}, Gladys Alfama1, Ghislaine Morvan Dubois1, Mathilde Paris4, Carine Giovannangeli2,3 and Barbara A. Demeneix1,*

1CNRS, UMR 5166, 7 rue Cuvier CP32 Paris Cedex 05, F-75231 France; MNHN, USM 501, Département de "Régulations, développement et diversité moléculaire", Evolution des Régulations Endocriniennes, 7 rue Cuvier CP32 Paris Cedex 05, F-75231 France, 2INSERM, U565, Acides nucléiques: dynamique, ciblage et fonctions biologiques, 57 rue Cuvier, CP26, Paris Cedex 05, F-75231, France, 3MNHN, USM503, Département de "Régulations, développement et diversité moléculaire", Laboratoire des Régulations et dynamique des génomes, 57 rue Cuvier, CP26, Paris Cedex 5, F-75231, France; CNRS, UMR5153, Acides nucléiques : dynamique, ciblage et fonctions biologiques, 57 rue Cuvier, CP26, Paris Cedex 5, F-75231, France and 4ENS Lyon, Laboratoire de Biologie Moléculaire de la Cellule, UMR CNRS 5161, 46 allée d’Italie, 69364 Lyon, France

*To whom correspondence should be addressed. Tel: +33 1 40 79 36 07; Fax: +33 1 40 79 36 18; Email: demeneix{at}mnhn.fr

Received July 26, 2006. Revised February 26, 2007. Accepted February 27, 2007.

RNA-interference-driven loss of function in specific tissues in vivo should permit analysis of gene function in temporally and spatially defined contexts. However, delivery of efficient short hairpin RNA (shRNA) to target tissues in vivo remains problematic. Here, we demonstrate that efficiency of polyethylenimine (PEI)-delivered shRNA depends on the regulatory sequences used, both in vivo and in vitro. When tested in vivo, silencing of a luciferase target gene by shRNA produced from a hybrid construct composed of the CMV enhancer/promoter placed immediately upstream of an H1 promoter (50%) exceeds that obtained with the H1 promoter alone (20%). In contrast, in NIH 3T3 cells, the H1 promoter was more efficient than the hybrid construct (75 versus 60% inhibition of target gene expression, respectively). To test CMV-H1 shRNA efficiency against an endogenous gene in vivo, we used shRNA against thyroid hormone receptor {alpha}1 (TR{alpha}1). When vectorized in the mouse brain, the hybrid construct strongly derepressed CyclinD1-luciferase reporter gene expression, CyclinD1 being a negatively regulated thyroid hormone target gene. We conclude that promoter choice affects shRNA efficiency distinctly in different in vitro and in vivo situations and that a hybrid CMV-H1 construct is optimal for shRNA delivery in the mouse brain.

{dagger}The authors wish it to be known that, in their opinion, the first and second authors contributed equally to this work.


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