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2007 Scientific Report

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VARI | 2007 Research Interests The Division of Quantitative Sciences includes the laboratories of Analytical, Cellular, and Molecular Microscopy (ACMM), the Laboratory of Microarray Technology (LMT), the Laboratory of Computational Biology, the Laboratory of Molecular Epidemiology, and the Laboratory of Mass Spectrometry and Proteomics. The Division’s laboratories use objective measures to define pathophysiologic events and processes. For example, the LMT measures the expression of genes relative to a control or a standard. When pathology and tissue organization is combined with expression, one can better determine not only what the change is but also possible causation, treatment targets, and effects of treatment. The Molecular Epidemiology laboratory builds objective data and pathology correlations to infer causation and prognosis. The ACMM laboratory has programs in pathology, histology, and imaging to describe and visualize changes in cell, tissue, or organ structure. Our imaging instruments allow us to visualize cells and their components with striking clarity, allowing researchers to determine where in a cell specific molecules are located. We also use a laser for microdissection of cells from a sample. The laboratory provides paraffin-block (SPIN program) and frozen-section (TAS program) staining of tissues. An archive of pathology tissues in the paraffin blocks (Van Andel Tissue Repository; VATR) is being accumulated with the cooperation of local hospitals, and the data on the samples is being converted to computerized files. The lab also carries out research that will improve our ability to quantify images, so that we will be able to not only state that a particular protein is present in an image, but also answer the questions of how much is there and with what other molecules is it co-localized? We are able to image using either fluorescent (e.g., FITC, GFP) or chromatic agents (e.g., DAB, H&E) and separate the components using our confocal, Nuance, or Maestro instruments. 47 The Laboratory of Microarray Technology provides gene expression analysis using cDNA microarrays. High-throughput robotics are used to maintain and process cDNA clone sets for the human, mouse, rat, and canine genomes. The clones are used to produce both cDNA and spotted oligonucleotide microarrays that are evaluated using strict quality control and quality assurance criteria developed using the Clinical Laboratory Improvement Amendments (CLIA) as a model. These criteria allow the laboratory to function in a manner consistent with fully accredited clinical laboratories. In 2006 we produced and used 790 cDNA microarrays, and we also produced 112 custom protein microarrays. In addition, the laboratory has expanded its services to include Agilent and Operon commercial oligonucleotide microarrays. The use of these products will remove much of the internal quality control and quality assurance burden, and they will also facilitate the requirement to perform array comparative genomic hybridization, chromatin immunoprecipitation (chip-on-chip), and splice variant analysis. Hauenstein Parkinson’s Center Throughout 2006 we have continued our collaboration with the Hauenstein Parkinson’s Center to collect patient blood samples and controls from 114 individuals. Mutations in the parkin gene in a series of families with more than one generation affected by Parkinson disease are being investigated by DNA sequence analysis and will be correlated to gene expression data obtained from microarray analysis.

Van Andel Research Institute | Scientific Report Blood spot arrays State laws in the U.S.A. mandate that blood be drawn from all newborn infants to screen for a variety of health-threatening conditions. The assays consume only a small portion of the blood samples, which are collected on filter paper (“Guthrie”) cards. Many states archive the leftover cards, often in unrefrigerated storage. Pete Haak and Eric Kort have successfully isolated mRNA from archived unfrozen neonatal blood spots obtained as long as nine years ago. Using both quantitative RT-PCR and multiplex gene expression analysis with cDNA arrays, we can detect RNA from hundreds to thousands of genes in these samples. Furthermore, we have shown through use of freshly spotted blood cards that the genes detected approximate those found in whole blood and purified buffy coat. These preliminary experiments demonstrate the feasibility of detecting and identifying RNA amplified from unfrozen stored neonatal blood spots. The application of high-throughput assays to the analysis of these widely available samples may be a valuable resource for the study of perinatal markers and determinants of subsequent disease development. The coming year will see this technology applied to the study of cerebral palsy and neuroblastoma. Mouse models of Parkinson disease 48 As part of the VAI initiative into Parkinson disease, we have begun to generate novel rodent models of dopaminergic cell loss in the brain in collaboration with Bart Williams. One of the key tools for these studies is the transgenic dopamine-transporter/cre (DAT-cre) mouse line, which specifically expresses the cre recombinase in dopaminergic neurons of the brain. In combination with other transgenic and knock-out mouse lines, the DAT-cre mice will allow us to address the response of such neurons to toxic stimuli in the context of specific gene deletions and additions. Several of the ongoing and future projects based on the DAT-cre mouse model are briefly described below. • Imaging and isolation of primary dopaminergic neurons from mouse brain. We have performed a genetic cross between the DAT-cre strain and ROSA26 reporter strain to generate mice that specifically express the LacZ reporter gene in dopaminergic neurons. The DAT-cre/ROSA26 mice will permit us to visualize and quantify live dopaminergic neurons in vivo. With these mice we will assess the effect of cytotoxic agents (e.g., MMTP, rotenone, or 6-hydroxydopamine) on the number of dopaminergic cells, and more importantly, assess the ability of mice to recover from these insults. These studies will provide insight into the regenerative capacity of the brain when dopaminergic neurons are lost or injured. The DAT-cre/ROSA26 mice will also provide a source of highly pure dopaminergic neurons for in vitro studies. Dopaminergic neurons from these mice will be isolated from brain tissue treated with DDAOgalactoside and will be identified from the cellular population by fluorescence-activated cell sorting in the VAI flow cytometry core facility. • Dopaminergic cell regeneration as a function of age. The relationship between age and the likelihood of developing Parkinson disease is well established, though the causal nature of this relationship is unclear. One hypothesis is that the capacity of the brain to regenerate damaged neurons decreases with age, consistent with a gradual loss of brain stem cells that give rise to new dopaminergic neurons. To test this hypothesis in a mammalian system, we are planning a genetic cross between DAT-cre and pu TK mice, the latter specifically expressing herpes simplex virus thymidine kinase (hsvTK) in cells that contain cre recombinase. Cells expressing hsvTK are sensitive to the antiviral compound ganciglovir (G418) and undergo programmed cell death after systemic treatment. Using the DAT-cre/pu TK model, we will eliminate dopaminergic neurons at various ages (3, 6, 9, and 12 months) and assess the regenerative potential of these mice using behavioral and histological parameters. These studies will indicate both the absolute and relative capacities of the mammalian brain to regenerate dopaminergic neurons as a function of age, thereby providing information about the value of therapies intended to stimulate the endogenous regenerative capacity of the brain in Parkinson disease patients.

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