| First Author | Gordeeva O | Year | 2018 |
| Journal | MGI Direct Data Submission | Mgi Jnum | J:262143 |
| Mgi Id | MGI:6161419 | Citation | Gordeeva O (2018) Expression of the genes of the melanoma antigen (Mage) families in male and female gonad. MGI Direct Data Submission |
| abstractText | Olga Gordeeva Kol'tsov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Street, Moscow, 119334, Russia Summary To understand an involvement of Mage (Melanoma antigen) genes in reproductive organ development the expression profiles of Mage gene families were studied in the testes and ovaries of C57Bl/6 mouse embryos at the E14.5 and E19.5 developmental stages and adult mice (P60 stage) using RT-PCR analysis. High expression of Mage-d1, -d2, -e1, -e2, -h1, -l2 genes (II class of Mages) was detected in the embryonic gonads of both sexes at all studied stages. However, gene expression of the Mage-a and Mage-b families (I class of Mages) varied between the testes and ovaries at studied stages. Mage-a2, -a4, -a6,-a8 and Mage-a10 were found to be expressed in embryonic gonads of both sexes at the E14.5 and E19.5 stages and adult testes, while no expression of Mage-a family genes was detected in adult ovaries. The expression of all Mage-b family genes was detected in embryonic and adult testes at all studied stages but no expression of Mage-b5 was detected in testes at the E19.5 stage. Mage-b3,-b4,-b16 and Mage-b18 but not Mage-b1 were expressed in the embryonic ovaries at the E14.5 and E19.5 stages. Weak expression of Mage-b5 was detected in embryonic ovaries. No expression of Mage-b family genes was detected in adult ovaries. Thus, extensive expression pattern of most genes of the Mage families detected in testes and ovaries of C57Bl/6 mouse embryos indicates wide involvement of these genes in the regulation of mouse germ line development. The expression patterns of I class of Mage genes varied during male and female gonad development while no differences were found in the expression patterns of II class of Mage genes in the embryonic gonads at studied stages. Methods RNA extraction and reverse transcription Testes and ovaries from the C57BL/6 mouse embryos at E 14.5, E19.5 and P60 stages were isolated and placed on ice. Total RNAs from the embryonic gonads (10 gonads of both sex at the E14.5 and E19.5 stages per sample, n=2; one testis and two ovaries at the P60 stage per sample, n=3) were extracted using the TRIzol® Reagent (Invitrogen). The RNA yield and quality were analyzed by NanoDrop 2000 (Thermo Scientific). Each sample was treated with TURBO DNase (Ambion/Invitrogen) according to the manufacturer's recommendations. cDNAs were synthesized using 1microM oligo-dT18 primer and 0.1 mM dNTP mix (Thermo Scientific/ Fermentas), 2 micrograms of total RNAs and 200U of reverse transcriptase SuperScript IV (Invitrogen). The expression of reference gene hypoxanthine guanine phosphoribosile transferase Hprt was used for normalization of cDNAs. Primer design and PCR PCR primers of 18-26 bp in length with a 40-60% GC content and Tm of 60 degrees C plus/minus 2 degrees C were selected using the Beacon Designer software (PREMIER Biosoft, USA) and PRIMER3 (Whitehead Institute/MIT Center for Genome Research, Cambridge, MA) to amplify products of 80-200 bp in length. Primers were pre-screened to determine the optimal conditions for specific cDNA amplification using adult mouse testes cDNAs as a positive control. They were tested using 30 PCR cycles at 60 degrees C annealing temperature under standard assay conditions (see below). PCR amplification Hot-lid PCR amplification of cDNA equivalent to 50 ng of total RNA was carried out in PCR mix containing 20 mM Tris-HCl buffer (pH 8.6 / 25°C) with 5 mM (NH4)2SO4, 20 mM KCl and 2.5 mM MgCl2, 0.25 U Maxima Hot Start Taq DNA polymerase (Thermo Scientific/Fermentas), 0.1 mM dNTPs (Thermo Scientific/Fermentas), and 0.2 microM of each primer were used at each reaction. PCR analysis of gene expression was carried out on an Eppendorf master cycler (Eppendorf, Germany) according to following program: preliminary denaturation at 95 degrees C for 5 min, each PCR cycle (35 cycles) consisted of 20 sec denaturing (95 degrees C), 20 sec annealing of primers (60 degrees C), and 30 sec elongation (72 degrees C) and final extension at 72°C for 10 min. All assays were conducted under identical conditions. The PCR products were then separated on a 1.5% agarose gel (TopVision Agarose, Fermentas) and Tris-borate buffer (x0.5, pH 8.3), stained with ethidium bromide and registered using transilluminator (BioRad, USA). |
