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Fluorescence-Based Laser Capture Microscopy Technology Facilitates Identification of Critical In Vivo Cytomegalovirus Transcriptional Programs

  • Craig N. Kreklywich
  • Patricia P. Smith
  • Carmen Baca Jones
  • Anda Cornea
  • Susan L. Orloff
  • Daniel N. StreblowEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1119)

Abstract

Cytomegalovirus gene expression in highly permissive, cultured fibroblasts occurs in three kinetic classes known as immediate early, early, and late. Infection of these cells results in a predictable transcriptional program leading to high levels of virus production. Infection of other, so-called, nonpermissive cell types results in a transcriptional program that either fails to produce virus particles or production is substantially reduced compared to fibroblasts. We have found that CMV gene expression profiles in tissues from infected hosts differ greatly from those observed in infected tissue culture cells. The number of viral genes expressed in tissues is much more limited, and the number of highly active genes does not correlate with viral DNA load. Additionally, viral gene expression in vivo is tissue selective with no two tissues expressing the exact same viral gene profile. Thus, in vivo CMV gene expression appears to be governed by mechanisms that are still uncharacterized. Cytomegalovirus remains in a persistent phase for the lifetime of the host. During this phase only a limited number of host cells are infected, and it is very difficult to detect CMV gene expression in whole tissues without sub-fractionating infected vs. uninfected cells. Herein, we describe the development of a fluorescence-based laser capture microscopy technique coupled with small sample size microarray analysis to determine the viral gene expression in 50–100 infected cells isolated from frozen RCMV-infected tissue sections.

Key words

Laser capture microscopy Cytomegalovirus Green fluorescence protein Microarray analysis 

Notes

Acknowledgements

The work presented in this manuscript was supported by grants from the National Institutes of Health (HL-083194 DNS) and (HL-66238-01 SLO). The Arcturus laser capture microscope used for this study was supported by Award Number S10 RR027503 from the National Center for Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. The Oregon National Primate Research Center Imaging Core is supported by NIH P51 RR000163. We thank Andrew Townsend for his assistance with graphics.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Craig N. Kreklywich
    • 1
    • 2
  • Patricia P. Smith
    • 1
  • Carmen Baca Jones
    • 1
  • Anda Cornea
    • 4
  • Susan L. Orloff
    • 1
    • 2
    • 3
  • Daniel N. Streblow
    • 1
    Email author
  1. 1.Department of Molecular Microbiology & ImmunologyVaccine & Gene Therapy Institute, Oregon Health & Science UniversityBeavertonUSA
  2. 2.Department of SurgeryOregon Health & Science UniversityPortlandUSA
  3. 3.Portland VA Medical CenterPortlandUSA
  4. 4.Oregon National Primate Research CenterOregon Health & Science UniversityBeavertonUSA

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