| First Author | Gordeeva O | Year | 2018 |
| Journal | MGI Direct Data Submission | Mgi Jnum | J:257298 |
| Mgi Id | MGI:6119627 | Citation | Gordeeva O (2018) Expression of the genes of the melanoma antigen (Mage) families in heart. 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 somatic organ development the expression profiles of Mage gene families was studied in the heart anlages of C57BL/6 mouse embryos at the E10.5, E14.5 and E19.5 developmental stages using the RT-PCR analysis. High expression of Mage-d1, -d2, -e1, -e2, -h1, -l2 genes (II class of Mages) was detected in the embryonic hearts at all studied stages. Expression of Mage-a and Mage-b family genes (I class of Mages) varied considerably between developmental stages. Mage-a2, -a4, -a6, -a8 and Mage-a10, were found to be expressed in the embryonic hearts at the E14.5 and E19.5 stages while expression of Mage-a2, -a4, -a6, and Marge-a8 was detected in the heart anlages at the E10.5 stage. The expression of Mage-b1, -b3, -b4, -b16, and -b18 was detected in the hearts at the E14.5 and E19.5 stages and Mage-b1, -b3, and -b16 was detected at the E10.5 stage. No expression of Mage-b5 and low expression of Mage-b4 was detected in the heart anlages at the developmental stages studied. Thus, the extensive expression pattern of most genes of the Mage families (16 of 17) detected in heart anlages of C57BL/6 mouse embryos indicates wide involvement of these genes in the regulation of mouse development. The expression patterns of I class Mage genes varied during heart development while no differences were found in the expression patterns of II class Mage genes in the embryonic hearts at studied stages. Methods RNA extraction and reverse transcription Heart from the C57BL/6 mouse embryos at E10.5, E 14.5 and E19.5 stages were isolated and placed on ice. Total RNAs from embryonic hearts (8 hearts at the E10.5 stage per sample, n=2; 3 hearts at the E14.5 stage per sample, n=3; 1 heart at the E19.5 stage per sample, n=3)) were extracted using the TRIzol Reagent (Invitrogen). Each sample was treated with Thermo Scientific dnDnase (Thermo Scientific/Fermentas) according to the manufacturer's recommendations. cDNAs were synthesized using 1 microM oligo-dT18 primer and 0.1 mM dNTP mix (Thermo Scientific/Fermentas), 1 microgram of total RNAs and 200U of Maxima Reverse Transcriptase (Thermo Scientific/Fermentas) according to the manufacturer's protocols. 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/Fermenras), 0.1 mM dNTPs (Thermo Scientific/Fermenras), 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 (37 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). |
