Reverse genetics in mammals has relied on gene targeting strategies and has mostly been limited to the mouse. Gene targeting through homologous recombination in mouse ES cells has drawbacks which include time, expense and complexity. Recently, a new approach has been developed based on RNA-interference (RNAi) which is simpler, faster, less expensive, and should be applicable to mammalian species other than mouse. The advent of RNAi is poised to accelerate the pace at which reverse genetics can be applied to study gene function in mammals.
"The method is particularly efficient in cells cultured in vitro, where interfering sequences can be either administered directly as transfected small interfering RNAs (siRNAs) or expressed as small hairpin RNAs (shRNAs), that are processed to siRNAs in the cell, using retroviral vectors. Transfer of shRNA producing constructs into mammalian embryos has been realized through pronuclear injection and transfection into embryonic stem cells (Xia et al., 2006). These methods suffer several limitations and a more promising method for large-scale application entails the use of lentiviral vectors, a gene delivery system that does not suffer developmental repression and that efficiently transduces embryos from many different species (Dann, 2007; Pfeifer, 2004). "
[Show abstract][Hide abstract] ABSTRACT: RNA interference (RNAi) through the use of lentiviral vectors is a valuable technique to induce loss of function mutations in mammals. Although very promising, the method has found only limited application and its general applicability remains to be established. Here we analyze how different factors influence RNAi mediated silencing of Col6a1, a gene of the extracellular matrix with a complex pattern of tissue specific expression. Our results, obtained with vectors pLVTHM and pLVPT-rtTRKRAB, point out three parameters as major determinants of the efficiency of interference: the choice of interfering sequence, the number of proviral copies integrated into the mouse genome and the site of insertion of the provirus. Although low copy number may produce efficient interference with low frequency, the general trend is that the number of integrated proviral copies determines the level of silencing and the severity of phenotypic traits. The site of insertion not only determines the overall intensity of expression of the small interfering RNA (siRNA), but also introduces slight variability of silencing in different organs. A lentiviral vector (pLVPT-rtTRKRAB) with doxycycline-inducible production of siRNA was also tested. Control of expression by the drug was stringent in many tissues; however, in some tissues turning off of siRNA synthesis was not complete. The data support the application of lentiviral vectors used here in transgenesis.
Journal of Biotechnology 05/2009; 141(1-2):8-17. DOI:10.1016/j.jbiotec.2009.02.013 · 2.87 Impact Factor
"RNAi is induced by short double-stranded RNA called small interfering RNA (siRNA), of which one strand (guide strand) is incorporated into RNA-Induced Silencing Complex (RISC) and directs localization and degradation of targeted RNA molecules with complementary sequence to the siRNA guide strand 4-6. Sustained RNAi can be achieved by introducing a gene-based construct that synthesizes short hairpin RNA (shRNA) from either RNA polymerase II or III (Pol II or III) promoters 2, 3. Pol III promoters such as U6 or H1 offer advantages over Pol II promoters, because the compact sequence of Pol III promoters is easier to handle and their ubiquitous expression pattern allows for broad silencing of a target gene 7-9. "
[Show abstract][Hide abstract] ABSTRACT: Transgenic RNAi, an alternative to the gene knockout approach, can induce hypomorphic phenotypes that resemble those of the gene knockout in mice. Conditional transgenic RNAi is an attractive choice of method for reverse genetics in vivo because it can achieve temporal and spatial silencing of targeted genes. Pol III promoters such as U6 are widely used to drive the expression of RNAi transgenes in animals. Tested in transgenic mice, a Cre-loxP inducible U6 promoter drove the broad expression of an shRNA against the Pink1 gene whose loss-of-functional mutations cause one form of familial Parkinson's disease. The expression of the shRNA was tightly regulated and, when induced, silenced the Pink1 gene product by more than 95% in mouse brain. However, these mice did not develop dopaminergic neurodegeneration, suggesting that silencing of the Pink1 gene expression from embryo in mice is insufficient to cause similar biochemical or morphological changes that are observed in Parkinson's disease. The results demonstrate that silencing of the PINK1 gene does not induce a reliable mouse model for Parkinson's disease, but that technically the inducible U6 promoter is useful for conditional RNAi in vivo.
International journal of biological sciences 03/2007; 3(4):242-50. · 4.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In vivo studies are an important tool for the identification and validation of novel drug targets in medicine; however, the interpretation of submitted and published data is often compromised by inadequate study design. Different study protocols, including the number of control groups and group size calculations, differ in target identification and validation studies. Furthermore, animal studies require that the selected target or compound meets the requirements for species specificity and target specificity; thus, providing the rationale for the selection of a particular species, strain, gender and age of the animals is necessary. Altogether, the presentation of target validation studies should meet defined criteria similar to those used in human trials.
Drug Discovery Today 07/2007; 12(11-12):446-51. DOI:10.1016/j.drudis.2007.04.001 · 6.69 Impact Factor
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