doi:10.1016/j.micpath.2006.05.003 
Copyright © 2006 Elsevier Ltd All rights reserved.
Short communication
Quantitative measurement of anthrax toxin receptor messenger RNA in primary mononuclear phagocytes
Christopher Premanandana, b, Michael D. Lairmorea, c, d, e, Soledad Fernandezf and Andrew J. Phippsa, b, 
, 
aDepartment of Veterinary Biosciences, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210-1093, USA
bThe Center for Microbial Interface Biology, The Ohio State University, Columbus OH 43210, USA
cComprehensive Cancer Center, The Arthur G. James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus OH 43210, USA
dDepartment of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus OH 43210, USA
eThe Center for Retrovirus Research, The Ohio State University, Columbus OH 43210, USA
fCenter for Biostatistics, The Ohio State University, Columbus OH 43210, USA
Received 8 February 2006;
revised 22 May 2006;
accepted 23 May 2006.
Available online 18 July 2006.
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Abstract
Two anthrax toxin receptors have been identified, tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). Both receptors have been shown to be capable of facilitating the entry of Bacillus anthracis exotoxins into the cytosol of susceptible cells. The levels of TEM8 and CMG2 RNA transcripts present in human primary macrophages derived from multiple unrelated donors and primary mouse macrophages have not been well described. In this communication, we examined the expression of mRNA transcripts of TEM8 and CMG2 in primary human and mouse macrophages and mouse tissues by standard and quantitative real-time RT-PCR. Our results indicate that CMG2 transcripts are preferentially expressed over TEM8 transcripts in primary human and mouse macrophages as compared to immortalized cell lines.
Keywords: Anthrax; TEM8; CMG2; Macrophage; Receptor
Fig. 1. Depiction of the predicted exons and introns for TEM8 and CMG2 showing alternative splicing patterns via dashed lines. The primers were designed to cross exon splice junctions that were common to all isoforms of TEM8 and CMG2 in addition to isoform-specific splice junctions for TEM8. Asterisks indicate the last exon in each isoform. †CMG2 isoform is a projected splice variant depicted in the Ensembl Genome Browser developed by the Sanger Institute and the European Bioinformatics Institute (Ensembl Gene ID ENSG00000163297). The sizes of the exons and introns are not drawn to scale.
Fig. 2. TEM8 isoform expression by standard RT-PCR. (A) Expression in a human cell line (HeLa) and human primary cells. (B) Expression in mouse cell lines, primary cells and tissues. Product size for the universal TEM8 is approximately 186 bp. Product size for human isoforms 1, 2 and 3 are 189, 122 and 130 bp, respectively. Purified PCR products were visualized by agarose gel electrophoresis. GAPDH expression was measured by RT-PCR as a positive amplification control in each experiment.
Fig. 3. mRNA expression of TEM8 and CMG2 by real-time RT-PCR. Results indicate the mean±SD. of three independent real-time RT-PCR reactions. Six human MDM donors were selected at random from the population of American Red Cross blood donors. ** represents below the level of detection (<1×104 copies/500 ng of RNA). *p<0.05 when compared with the same receptor expression in Hela cells (non-parametric Kruskal–Wallis test). Comparison of CMG2 expression in donors was performed using one-way analysis of variance (ANOVA). Differences in CMG2 expression between donors was insignificant (p>0.5).
Table 1.
RT-PCR primer sequences and expected amplicon size
a National Center for Biotechnology Information (NCBI), National Library of Medicine, National Institutes of Health accession numbers.
b β2M—beta-2-microglobulin.
c GAPDH—glyceraldehyde-3-phosphate dehydrogenase.
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