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 Orloff, Daniel Streblow

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)217-237
Number of pages21
JournalMethods in Molecular Biology
Volume1119
DOIs
StatePublished - 2014
Externally publishedYes

Fingerprint

Cytomegalovirus
Confocal Microscopy
Fluorescence
Technology
Viral Genes
Gene Expression
Fibroblasts
Viral DNA
Microarray Analysis
Infection
Viral Load
Transcriptome
Virion
Sample Size
Cell Culture Techniques
Cell Count
Viruses
Genes

Keywords

  • Cytomegalovirus
  • Green fluorescence protein
  • Laser capture microscopy
  • Microarray Analysis

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Medicine(all)

Cite this

Fluorescence-based laser capture microscopy technology facilitates identification of critical in vivo cytomegalovirus transcriptional programs. / Kreklywich, Craig N.; Smith, Patricia P.; Jones, Carmen Baca; Cornea, Anda; Orloff, Susan; Streblow, Daniel.

In: Methods in Molecular Biology, Vol. 1119, 2014, p. 217-237.

Research output: Contribution to journalArticle

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