BioProject

In order to study the microglia contribution in neurodegeneration more specifically we established a mouse model of prion disease in which the 79A murine prion strain was introduced by an intraperitoneal route into BALB/cJFms-EGFP/- mice, which express Enhanced Green Fluorescent Protein (EGFP) under control of the C-fms operon. More...

In order to study the microglia contribution in neurodegeneration more specifically we established a mouse model of prion disease in which the 79A murine prion strain was introduced by an intraperitoneal route into BALB/cJFms-EGFP/- mice, which express Enhanced Green Fluorescent Protein (EGFP) under control of the C-fms operon.  Samples were taken at time points during disease progression and histological analysis of the brain and transcriptional analysis of isolated microglia was carried out. The analysis of isolated microglia revealed a disease specific, highly pro-inflammatory signature in addition to an up-regulation of genes associated with metabolism, respiratory stress and DNA repair. This study strongly supports the growing recognition of the importance of microglia within the prion disease process and identifies the nature of the response through gene expression analysis of isolated microglia. Overall design: Groups of gender matched BALB/cJFms-EGFP/- mice were challenged i.p. with either 0.02 ml of 0.02 w/v 79A infected or normal brain material. The average initial age of animals at challenge was 116 days ± 2.1 SE. Mice were culled in duplicate at 35, 100, 150 and 200 dpi. Microglial RNA processing was handled by ARK Genomics (The Roslin Institute & R(D)SVS). The mRNA was converted to double-stranded cDNA containing biotin, using a single round of amplification, from both the poly-A 3 ́ tail and randomly using the NuGen Ovation® picoSL WTA system (NuGen, 3310) followed by the NuGen Encore® Biotin module biotin labelling process (NuGen, 4200). The cDNA samples were hybridised to Affymetrix Mouse Gene 1.1 arrays on an Affymetrix GeneTitan® Multi-Channel instrument (Affymetrix, High Wycombe, UK) incorporating the hybridisation, washing, staining and scanning stages. Annotation and quality control was performed with the Affymetrix Expression Console. Normalisation was undertaken using the robust multi-array average expression measure (Irizarry et al., 2003). BioLayout Express3D was chosen for analysis of this dataset. The software has the ability to accommodate very large gene datasets which are viewed in a three dimensional neighbourhood with other genes based on a user defined threshold of co-expression. The capacity to handle very large datasets is integral to the software design in which the optimized graph layout and clustering algorithm are rendered within a hardware-accelerated OpenGL framework using the computer graphics processing unit and its inherent capability to handle very large numbers of calculations in a short period of time (Freeman et al., 2007). Less...