BioProject

Werner syndrome (WS) is a rare disorder characterized by the premature onset of a number of age-related diseases. More...

Werner syndrome (WS) is a rare disorder characterized by the premature onset of a number of age-related diseases. The gene responsible for WS is believed to be involved in different aspects of transcription, replication, and/or DNA repair. The poly(ADP-ribose) polymerase-1 (PARP-1) enzyme is also involved in DNA repair and is known to affect transcription of several genes. In this study, we examined the expression profile of cells lacking the normal function of either or both enzymes. All mutant cells exhibited altered expression of genes normally responding to oxidative stress. Interestingly, more than 58% of misregulated genes identified in double mutant cells were not altered in cells with either the Wrn or PARP-1 mutation alone. Consequently, the impact on gene expression profile when both Wrn and PARP-1 are mutated was greater than a simple addition of individual mutant genotype. In addition, double mutant cultured cells showed major misregulation of genes involved in apoptosis, cell cycle control, embryonic development, metabolism, and signal transduction. More importantly, in vivo analyses of double mutant mice have confirmed the increased apoptosis and the developmental defects in embryos as well as the major increase in intracellular phosphorylation and oxidative DNA damage in adult tissues. They also exhibited a progressive increase in oxidative stress with age. Thus, a major result of this study is that changes in expression of several genes and physiological functions identified in vitro were confirmed in mouse embryonic and adult tissues. Keywords: Genetic modification Overall design: Microarray analyses were performed on cell cultures at the second passage (10 population doublings). For each genotype, asynchronously dividing cells derived from three healthy 15.5 day embryos from one litter were pooled at the second passage and cytoplasmic RNA was extracted. Cytoplasmic RNA was used in these experiments to avoid contamination with heterogeneous nuclear RNA and genomic DNA. (Note that DNAse treatment was also applied to all samples). This pool of RNA was labeled sample number 1 for each genotype. Pooling of embryonic cells was performed to minimize the effect of inter-individual biological differences. A second pool of embryonic cells was also created from a separate dam (second litter) for each genotype (called samples number 2). This strategy allowed obtaining samples in duplicate for each genotype. The cRNA from wild type cells were synthesized with Cy-5 labeled nucleotides and cRNAs from Wrn mutant, PARP-1 null, and PARP-1 null/Wrn mutant cells were synthesized with Cy-3 labeled nucleotides. Hybridization was performed on Mouse Agilent 60-mer Oligo Microarray chips by mixing wild type labeled cRNA (baseline expression levels) with either Wrn mutant, PARP-1 null, or PARP-1 null/Wrn mutant cRNA. Hybridization experiments were done in duplicates. Less...