Adenovirus-delivered antisense RNA and shRNA exhibit different silencing efficiencies for the endogenous transforming growth factor-beta (TGF-beta) type II receptor.
Gene silencing is an essential tool in gene discovery and gene therapy. Traditionally, viral delivery of antisense RNA and, more recently, small interfering RNA (siRNA) molecules in the form of small hairpin RNAs (shRNA) has been used as a strategy to achieve gene silencing. Nevertheless, the enduring challenge is to identify molecules that specifically and optimally silence a given target gene. In this study, we tested a set of adenovirus-delivered antisense RNA fragments and adenovirus-delivered shRNA molecules for their ability to target human transforming growth factor-beta type II receptor (TGFbetaRII). We used a dicistronic reporter, consisting of the coding sequences for TGFbetaRII and green fluorescent protein (GFP) to screen for optimal silencing agents targeting TGFbetaRII. Our results show, for both antisense RNA and shRNA molecules, that their effectiveness in the GFP screen correlated directly with their ability to reduce exogenously expressed TGFbetaRII. Unexpectedly, the antisense RNAs were unable to silence endogenous TGFbetaRII. In contrast, the shRNAs were able to silence endogenous TGFbetaRII. The shRNA that demonstrated the most pronounced effect on the dicistronic TGFbetaRII/GFP reporter reduced endogenous TGFbetaRII protein expression by 70% in A549 cells and reduced TGFbeta signaling by >80% in HeLa cells.
Biotechnology Research Institute, Montréal, Québec, Canada H4P 2R2.
Miroslava
M
Ogorelkova
John
J
Zwaagstra
Seyyed Mehdy
SM
Elahi
Christel
C
Dias
Claire
C
Guilbaut
Rita
R
Lo
Cathy
C
Collins
Maria
M
Jaramillo
Alaka
A
Mullick
Maureen
M
O'Connor-McCourt
Bernard
B
Massie
D000256:Adenoviridae / Q000235:genetics; D015536:Down-Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D017346:Protein Serine-Threonine Kinases; D034622:RNA Interference; D016372:RNA, Antisense / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000235:genetics; D014162:Transfection
D016372:RNA, Antisense; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II