BACKGROUND: Viral vectors have been used in several different settings for the delivery of small hairpin (sh) RNAs. However, most vectors have utilized ubiquitously-expressing polymerase (pol) III promoters to drive expression of the hairpin as a result of the strict requirement for precise transcriptional initiation and termination. Recently, pol II promoters have been used to construct vectors for RNA interference (RNAi). By embedding the shRNA into a micro RNA-context (miRNA) the endogenous miRNA processing machinery is exploited to achieve the mature synthetic miRNA (smiRNA), thereby expanding the possible promoter choices and eventually allowing cell type specific down-regulation of target genes. METHODS: In the present study, we constructed lentiviral vectors expressing smiRNAs under the control of pol II promoters to knockdown gene expression in cell culture and in the brain. RESULTS: We demonstrate robust knockdown of green fluorescent protein using lentiviral vectors driving RNAi from the ubiquitously-expressing promoter of the cytomegalovirus (CMV) and, in addition, we show for the first time neuron-specific knockdown in the brain using a neuron-specific promoter. Furthermore, we show that the expression pattern of the presumed ubiquitously-expressing CMV promoter changes over time from being expressed initially in neurons and glial cells to being expressed almost exclusively in neurons in later stages. CONCLUSIONS: In the present study, we developed vectors for cell-specific RNAi for use in the brain. This offers the possibility of specifically targeting RNAi to a subset of cells in a complex tissue and may prove to be of great importance in the design of future gene therapeutic paradigms. 2009 John Wiley & Sons, Ltd.
BACKGROUND: Viral vectors have been used in several different settings for the delivery of small hairpin (sh) RNAs. However, most vectors have utilized ubiquitously-expressing polymerase (pol) III promoters to drive expression of the hairpin as a result of the strict requirement for precise transcriptional initiation and termination. Recently, pol II promoters have been used to construct vectors for RNA interference (RNAi). By embedding the shRNA into a micro RNA-context (miRNA) the endogenous miRNA processing machinery is exploited to achieve the mature synthetic miRNA (smiRNA), thereby expanding the possible promoter choices and eventually allowing cell type specific down-regulation of target genes. METHODS: In the present study, we constructed lentiviral vectors expressing smiRNAs under the control of pol II promoters to knockdown gene expression in cell culture and in the brain. RESULTS: We demonstrate robust knockdown of green fluorescent protein using lentiviral vectors driving RNAi from the ubiquitously-expressing promoter of the cytomegalovirus (CMV) and, in addition, we show for the first time neuron-specific knockdown in the brain using a neuron-specific promoter. Furthermore, we show that the expression pattern of the presumed ubiquitously-expressing CMV promoter changes over time from being expressed initially in neurons and glial cells to being expressed almost exclusively in neurons in later stages. CONCLUSIONS: In the present study, we developed vectors for cell-specific RNAi for use in the brain. This offers the possibility of specifically targeting RNAi to a subset of cells in a complex tissue and may prove to be of great importance in the design of future gene therapeutic paradigms. 2009 John Wiley & Sons, Ltd.
Authors: Thomas H Hutson; Edmund Foster; John M Dawes; Robert Hindges; Rafael J Yáñez-Muñoz; Lawrence D F Moon Journal: J Gene Med Date: 2012-05 Impact factor: 4.565
Authors: Marijke W A de Backer; Carlos P Fitzsimons; Maike A D Brans; Mieneke C M Luijendijk; Keith M Garner; Erno Vreugdenhil; Roger A H Adan Journal: BMC Neurosci Date: 2010-07-13 Impact factor: 3.288
Authors: Madeleine V King; Nisha Kurian; Si Qin; Nektaria Papadopoulou; Ben H C Westerink; Thomas I Cremers; Mark P Epping-Jordan; Emmanuel Le Poul; David E Ray; Kevin C F Fone; David A Kendall; Charles A Marsden; Tyson V Sharp Journal: Neuropsychopharmacology Date: 2013-08-28 Impact factor: 7.853