Studies in this application are designed to use DNA microarray technology to attain a comprehensive understanding of gene expression in differentiating cytotrophoblasts (CTBs) in the placenta and to assess the impact of human cytomegalovirus (CMV) infection on expression pathways in these cells. We seek to define a new biology of CMV pathogenesis by identifying viral genes that dysregulate the differentiation of CTBs during placental development. Through a collaboration with the laboratory of Dr. Joseph DeRisi, a pioneer in DNA microarray technology, we propose to broaden the scope of the parent grant (AI46657) substantially. Our plan is to use human and CMV genomic arrays to explore gene expression pathways in CTBs before and after CMV infection. This approach will allow interrogation of the entire transcriptional program without requiring an a priori choice of which genes to study. Thus, there is a very high likelihood of discovering important changes in the expression of novel or unexpected genes, which would be very difficult without the global assessment made possible by this new technology. Dr. DeRisi has organized a microarray core facility for genomics and proteomics at UCSF that offers equipment, protocols, software, and technical training in microarray printing, hybridization, and data analysis. With assistance from the Dr. DeRisi laboratory, we will examine patterns of cellular and viral gene expression simultaneously on microarrays containing 70-mer oligonucleotides representing 14,000 human genes and the entire CMV genome.
Our specific aims are (1) to determine patterns of cellular gene expression in differentiating CTBs and investigate the impact of CMV infection with laboratory and pathogenic strains and (2) to identify CMV genes that affect CTB expression pathways. Changes in expression suggested in microarray experiments will be verified independently by analysis of protein and RNA levels and functional assays with support of the parent grant. The expertise and resources available through this collaboration will enable us to achieve a global view of expression pathways associated with CTB differentiation and immune functions and to deepen our understanding of CMV pathogenesis in a clinically relevant primary cell type. Gene exploration facilitated by microarray technology could reveal novel targets for molecular diagnosis and lead to therapies that block prenatal CMV infection. Similarities between expression pathways associated with invasion and angiogenesis in CTBs and tumorigenic cells suggest the added potential of these studies to identify novel targets for therapies and CMV genes that could be used to treat tumor progression in cancer.
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