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ABSTRACT: We silenced p53 gene expression in ARPE-19, a human retinal pigmented epithelial cell line using RNA interference. The effect of silencing the p53 gene in proliferating ARPE-19 cells was studied. Four short hairpin RNAs (shRNAs) targeting different regions of human p53 mRNA were delivered individually into ARPE-19 cells using lentiviral vector to produce stable cell lines. p53 mRNA and protein levels were reduced to varying extents in the four shRNA-transduced ARPE-19 cell lines. The cell line that showed greatest reduction (85-90%) of p53 expression showed decreased p21 promoter activation after DNA damage with camptothecin, etoposide and MMS. Whereas treatment of wild type ARPE-19 cells with camptothecin resulted in apoptosis, silencing p53 expression increased their survival. Cell cycle analyses indicated that irradiation resulted in a G(1) arrest in ARPE-19 cells, and that the arrest was significantly reduced in p53-silenced cells. Thus, p53 plays a central role in the response of ARPE-19 cells to DNA damaging agents that act via different mechanisms. Additionally, ARPE-19 cells with reduced p53 expression behave similar to tumor cell lines with mutated or non-functional p53. The present data demonstrate the utility of lentiviral vectors to create stable isogenic cell lines with reduced expression of a specific gene, thereby permitting the study of the function of a gene, the pathways controlled by it, and the effect of therapeutics on a cell with altered genetic makeup in a pair-wise fashion.
Cell cycle (Georgetown, Tex.) 06/2005; 4(5):697-703. · 5.36 Impact Factor
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ABSTRACT: Lentiviral vectors are efficient tools for the introduction of genes into a wide range of established and primary cells in vitro, ex vivo, and in vivo, and also permit efficient transgenesis in a wide range of mammalian species. Our goals have been to apply the broad capabilities of the lentiviral vector system to AD research. Using a set of vectors expressing APP and PS1 genes, we demonstrated the efficiency and fidelity of the system for in vitro biochemical analyses of genes and pathways involved in plaque deposition. These analyses were performed in cell lines and in primary neuronal cultures, which have previously been difficult to use. The methods and tools described here are applicable to the study of effects of other genes and gene combinations on APP processing, including suppression of gene activity by delivering shRNAs. We have attempted to create local plaque pathology by stereotactic injection of APP and PS1 expressing vectors into mouse brains for use as a rapid model for plaque pathology that can be used in a broad range of mammals. No amyloid or preamyloid pathology has been detected over a six-month period; the possible reasons are discussed. Lastly, we have used the vectors to create transgenic rats expressing mutant APP and mutant PS1 and have obtained the first set of positive pups with more expected. The results presented here demonstrate the utility of Lentiviral vector-based approaches to the study of AD and other neurodegenerative diseases.
Current Alzheimer Research 05/2005; 2(2):239-47. · 3.95 Impact Factor
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ABSTRACT: Development of therapeutics for Alzheimer's disease (AD) requires appropriate cell culture models that reflect the errant biochemical pathways and animal models that reflect the pathological hallmarks of the disease as well as the clinical manifestations. In the past two decades AD research has benefited significantly from the use of genetically engineered cell lines expressing components of the amyloid-generating pathway, as well as from the study of transgenic mice that develop the pathological hallmarks of the disease, mainly neuritic plaques. The choice of certain cell types and the choice of mouse as the model organism have been mandated by the feasibility of introduction and expression of foreign genes into these model systems. We describe a universal and efficient gene-delivery system, using lentiviral vectors, that permits the development of relevant cell biological systems using neuronal cells, including primary neurons and animal models in mammalian species best suited for the study of AD. In addition, lentiviral gene delivery provides avenues for creation of novel models by direct and prolonged expression of genes in the brain in any vertebrate animal. TranzVector is a lentiviral vector optimized for efficiency and safety that delivers genes to cells in culture, in tissue explants, and in live animals regardless of the dividing or differentiated status of the cells. Genes can also be delivered efficiently to fertilized single-cell-stage embryos of a wide range of mammalian species, broadening the range of the model organism (from rats to nonhuman primates) for the study of disease mechanism as well as for development of therapeutics.
Journal of Molecular Neuroscience 02/2004; 24(1):23-32. · 2.50 Impact Factor
