Adenovirus-mediated delivery of short hairpin RNA (shRNA) mediates efficient gene silencing in terminally differentiated cardiac myocytes.
RNA interference (RNAi) represents the most frequently utilized technique to analyze proteins by loss of function assays. Protein synthesis is impaired by sequence-specific degradation of mRNA, which is triggered by short (19-28 nt) silencing RNAs (siRNA). Efficient gene silencing using RNAi has been demonstrated in numerous cell lines and primary cultured cells. Incorporation of siRNA into terminally differentiated mammalian cells, such as adult cardiac myocytes is limited by their resistance to standard transfection protocols. Viral delivery of short-hairpin RNA (shRNA) overcomes these limitations and allows efficient gene silencing in these cells. This chapter describes the generation and characterization of recombinant siRNA-encoding adenoviruses and their application to adult cardiac myocytes, which represent a standard experimental model in research related to cardiac physiology and pathophysiology. Feasibility of this approach is demonstrated by effective ablation (>80%) of both, a transgene encoding for eGFP and the endogenous muscarinic M(2) acetylcholine receptor.
Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA. andreas.rinne@rub.de
Andreas
A
Rinne
Christoph
C
Littwitz
Kirsten
K
Bender
Marie-Cécile
MC
Kienitz
Lutz
L
Pott
D000256:Adenoviridae / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism*; D034622:RNA Interference; D051381:Rats; D014771:Virion / Q000235:genetics / Q000302:isolation & purification