Immune-related gene expression profile in laboratory common marmosets assessed by an accurate quantitative real-time PCR using selected reference genes

PLoS One. 2013;8(2):e56296. doi: 10.1371/journal.pone.0056296. Epub 2013 Feb 25.

Abstract

The common marmoset (Callithrix jacchus) is considered a novel experimental animal model of non-human primates. However, due to antibody unavailability, immunological and pathological studies have not been adequately conducted in various disease models of common marmoset. Quantitative real-time PCR (qPCR) is a powerful tool to examine gene expression levels. Recent reports have shown that selection of internal reference housekeeping genes are required for accurate normalization of gene expression. To develop a reliable qPCR method in common marmoset, we used geNorm applets to evaluate the expression stability of eight candidate reference genes (GAPDH, ACTB, rRNA, B2M, UBC, HPRT, SDHA and TBP) in various tissues from laboratory common marmosets. geNorm analysis showed that GAPDH, ACTB, SDHA and TBP were generally ranked high in stability followed by UBC. In contrast, HPRT, rRNA and B2M exhibited lower expression stability than other genes in most tissues analyzed. Furthermore, by using the improved qPCR with selected reference genes, we analyzed the expression levels of CD antigens (CD3ε, CD4, CD8α and CD20) and cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12β, IL-13, IFN-γ and TNF-α) in peripheral blood leukocytes and compared them between common marmosets and humans. The expression levels of CD4 and IL-4 were lower in common marmosets than in humans whereas those of IL-10, IL-12β and IFN-γ were higher in the common marmoset. The ratio of Th1-related gene expression level to that of Th2-related genes was inverted in common marmosets. We confirmed the inverted ratio of CD4 to CD8 in common marmosets by flow cytometric analysis. Therefore, the difference in Th1/Th2 balance between common marmosets and humans may affect host defense and/or disease susceptibility, which should be carefully considered when using common marmoset as an experimental model for biomedical research.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Callithrix / genetics*
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / genetics
  • Interleukin-10 / genetics
  • Interleukin-13 / genetics
  • Interleukin-2 / genetics
  • Interleukin-4 / genetics
  • Interleukin-5 / genetics
  • Interleukin-6 / genetics
  • Real-Time Polymerase Chain Reaction
  • Transcriptome / genetics*
  • Tumor Necrosis Factor-alpha / genetics

Substances

  • Interleukin-13
  • Interleukin-2
  • Interleukin-5
  • Interleukin-6
  • Tumor Necrosis Factor-alpha
  • Interleukin-10
  • Interleukin-4
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)

Grants and funding

This work was supported in part by Grants-in-Aid for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labor, and Welfare, Japan (Grants H20-shinkou-ippan-013 and H23-shinkou-ippan-010) as well as by Grant-in-Aid for Challenging Exploratory Research 23659237 from the Japan Society for the Promotion of Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.