• Introduction
  • Methods
  • Data Analysis

     The melanoma antigen (MAGE) protein family is conserved in all eukaryotes, but has rapidly expanded in mammals and currently consists of more than 37 genes in human and mouse. MAGEs have been intensively pursued as potential targets for therapeutic cancer vaccines, but what they normally do remains largely unexplored. We have recently begun to understand their molecular function in posttranslational modification of proteins by ubiquitination. To uncover their biological role, we first determined the precise location and timing of expression of all MAGE genes in human and mouse. We used quantitative real time PCR to generate a comparative anatomical expression atlas of all MAGE genes in the adult human and mouse tissues. Further, we determined their expression during embryonic development of the mouse and during first wave of spermatogenesis/folliculogenesis in the testis and ovary. Our findings uncover unanticipated insights into the physiological role of MAGEs in germ cells. This MAGE gene expression atlas provides the first systematic analysis of MAGE gene expression in the human and mouse and offers novel insights into their function during spermatogenesis.

Data for this study originated at and is used with permission from UT Southwestern

Methods

     Tissues were obtained from commercially available sources (human) or collected from C57BL/6 or BALB/c mice. Except for female-reproductive tissues, all tissues were from male mice. For embryonic tissue panel, ICR mouse embryos were taken at E15, E16, E17, E18, and E19. Placenta and fetal organs were collected (heart, lung, brain, adrenal, liver, kidney, colon, and gonads. Except for ovary, all other organs were from male embryos. For the first wave of spermatogenesis/folliculogenesis panel, testes and ovaries were collected from C57BL/6 mice at P5, P7, P10, P14, P21, P28, P35, P42, and P56. Populations of cells highly enriched for specific spermatogenic cell types were prepared from C57BL/6 mice. RNA was extracted using RNA Stat 60 reagent. Total RNA was pooled in equal quantities for each tissue (n = 6). Genomic DNA contamination was eliminated by DNase I (Roche) treatment and cDNA for RT-QPCR assays was prepared from 4 μg DNased RNA using High Capacity cDNA Reverse Transcription kit (Life Technologies) in 100 μL final volume. Following cDNA synthesis, RNAse-free water was added to increase the sample volume to 300 μL. Gene expression levels were measured by RT-QPCR in triplicate wells of a 384-well reaction plate with 10 ng cDNA per well on an Applied Biosystems 7900HT with SYBR Green chemistry. Normalized mRNA levels are expressed as arbitrary units and were obtained by dividing the averaged, efficiency corrected values for mRNA expression by that for 18s rRNA and multiplied by 1x105 for scaling purposes.

Anatomical MAGE gene expression in C57BL/6 mouse

Anatomical MAGE gene expression in BALB/c mouse

MAGE gene expression during embryonic development of the mouse

MAGE gene expression during first wave of spermatogenesis and folliculogenesis in testis and ovary

MAGE gene expression in enriched fractions of specific testis cell types

MAGE gene expression in mouse embryonic stem cells

MAGE gene expression in the testis of steel mice (KitlSl/KitlSl-d)

Anatomical MAGE gene expression in human tissues

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