| First Author | Gordeeva O | Year | 2018 |
| Journal | MGI Direct Data Submission | Mgi Jnum | J:260954 |
| Mgi Id | MGI:6154134 | Citation | Gordeeva O (2018) Expression Patterns of Cancer-Testis Antigens of Mage Families in Somatic and Reproductive Organs of Immunocompetent and Immunodeficient Mice. 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 functions of somatic and reproductive organs of mice with different genetic and immunological backgrounds, the expression profiles of Mage gene families were studied in the brain, heart, liver, ovary and testis of adult immunocompetent and immunodeficient mice of C57Bl/6 and BALB/cOlaHsd-Foxn1nu strains, respectively. No significant differences were detected in the expression of Mage genes examined in the organs of mice with different genetic and immunological backgrounds. High expression of Mage-d1, -d2, -e1, -e2, -h1, -l2 genes (II class of Mages) was detected in somatic and reproductive organs of adult immunocompetent and immunodeficient mice. The expression of 10 studied genes of Mage-a and Mage-b family genes (I class of Mages) was detected only in the testes of both mouse strains. Thus, these findings show that the expression pattern of studied genes of Mage families in reproductive and somatic organs is independent of the genetic or immunological backgrounds in mice. Methods RNA extraction and reverse transcription Organ samples from the C57BL/6 and immunodeficient BALB/cOlaHsd-Foxn1nu mice in age of 75 days age were isolated and placed on ice. Total RNAs were extracted using the TRIzol® Reagent (Invitrogen). For analysis total RNAs were isolated from 20-30 mg of tissues (n=3 samples). 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/Fermenras), 1 micrograms of total RNAs and 200U of Maxima Reverse Transcriptase (Thermo Scientific/Fermenras) according to the manufacture'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 100 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 (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). |
