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The mouse Polyomavirus infection in non-ciliated bronchiolar epithelial (Clara) cells provides an in vivo system to examine the genetic factors facilitating viral replication and under specific conditions, tumorgenesis. Currently little is known about how or in what way the virus alters the host cell environment to favor replication and avoid host antiviral response. We present a novel approach to studying Polyomavirus (Py) with the potential to analyze the genetic changes that promote cell proliferation and in turn sustaining viral replication. In Py infected cells, the Clara Cell Secretory Protein (CCSP) gene that decreases the lung inflammatory response to acute viral infection is down regulated, while the Proliferating Cell Nuclear Antigen (PCNA) gene that is required for chromosomal DNA replication is upregulated. In this study, immunohistochemical staining of Py infected mouse lung sections was optimized for CCSP and PCNA, thus enabling us to differentiate infected from non-infected Clara cells. Based on the different staining patterns, we were able to separately collect infected and non-infected Clara cells using Laser Capture Microdissection (LCM). To analyze host gene regulation differences between infected and non-infected Clara cells, RNA was isolated from these cell groups and linearly amplified. Preliminary gene analysis consists of using micro arrays to characterize the gene profiles in the infected cells compared to that of the non-infected cells. This study can provide basic insight regarding modification of cellular gene expression in Clara cells promoting viral replication and a predisposition to tumor genesis in response to Py infection.
The Polyomavirinae are small non-enveloped double-stranded DNA viruses with icosahedral capsids. Since their discovery in 1953, twelve types of polyomaviruses have been identified so far. Of these, SV40 in primates, BKV and JCV in humans and Py in mice have been primarily studied. The mouse polyomavirus system is used to study tumor induction and cell specific DNA regulation. Under laboratory conditions, inoculated newborn mice develop a high frequency of mammary gland, salivary gland, kidney, and thymic tumors as a secondary infection. Before reaching these sites of persistent infection, the polyomavirus must establish a primary infection in the lung. Within the lung, Py infection is restricted to non-ciliated bronchiolar epithelial (Clara) cells. Previous studies have focused on specific genes encoding tumor-inducing antigens, such as large and middle T-antigens rather than examining the multitude of genes involved in tumorgenesis. In this study we have combined the modern technology of Laser Capture Microdissection (LCM) with the technology of genetic analysis using high-density cDNA microarray chips. By selecting specific cells from tissue sections with LCM and linearly amplifying RNA extracted from these cells, we aim to compare the different gene expression profiles of Py infected and uninfected cells. This approach combined with eventual proteonomic analysis will produce a more thorough understanding of how Py modifies host cells in vivo to favor viral replication and in some cases tumorgenesis.
Immunohistochemistry: Intranasal Py infected lung tissue from SCID mice was obtained from K. Gottlieb. Tissues were fixed, dehydrated, embedded in paraffin blocks and sectioned for Immunohistochemical staining. Monoclonal antibodies against CCSP (K. Gottlieb) and PCNA (Sigma-Aldrich, Inc.) were used. A biotinylated anti-mouse secondary antibody conjugated to streptavidin peroxidase was used for the PCNA antibodies. A biotinylated anti-goat secondary antibody conjugated to avidin-peroxidase (Vector Laboratories) was used for the CCSP antibodies. Staining was performed with DAB (3-3’-diaminobenzidine).
Laser Capture Microdissection: The PixCell II LCM system (Acturus Engineering) was used to microdissect infected and non-infected Clara cells from dehydrated stained slides. Approximately 200-250 CCSP positive stained cells were captured with a 7.5-µm laser beam. The capturing criteria for a “stained” cell consisted of still being able to distinguish the dark brown staining when the microscope light intensity was adjusted to the highest setting.
- Infected and non-infected Clara cells can be distinguished using CCSP and PCNA primary antibodies.
- Specific cell populations can be captured using Laser Capture Microscopy in conjunction with immunohistochemical staining.
- Preliminary data indicates sufficient RNA can be extracted from 200-500 cells captured under LCM.
We thank Nima Farsinejad and Youkyung Choi for their technical assistance. This work was supported the Howard Hughes Medical Institute Grant No. 5200307 and the Cardile Foundation Grant (CH), NIH Grant No. DK56381 (CH), CA63640 (CH).
Jason combined immunohistochemical staining with Laser Capture Microdissection (LCM) technology to evaluate the different in vivo gene expression profiles between polyomavirus infected and non-infected Clara cells from mouse. Using the specific staining patterns, he was able to separately isolate infected and non-infected cells. The RNA was then isolated from these two cell groups and linearly amplified. This RNA product was then used on microarray chips allowing him to examine the plethora of genes that are upregulated or downregulated. The in vivo gene profile differences can be compared to examine those genes which might be key to tumorgenesis and/or viral survival.
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