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The ovine genome contains at least 20 copies of endogenous beta retroviruses (enJSRVs) that are highly homologous with Jaagsiekte sheep retrovirus (exJSRV), an exogenous retrovirus which is responsible for ovine pulmonary adenocarcinoma (OPA)[1, 7, 8, 13, 27]. This infectious tumor naturally involves two types of secretary epithelial cells of the lung, alveolar typeⅡcells and non-ciliated bronchiolar epithelial cells[15, 17]. The cellular receptor for both exJSRV and the enJSRV is hyaluronidase 2 (Hyal-2) and JSRV interacts with target cells by competing the membrane receptor with enJSRV[19, 25]. The relationship between exJSRV and enJSRV is still unclear. It is assumed that endogenous retroviruses (ERVs) are derived from integration events during evolution of ancient exogenous retroviruses into the germline of host animal species. ERVs are transmitted through the germline as stable Mendelian genes, and their structure and sequence are similar to infectious exogenous retroviruses. Some study reports revealed that JSRV genomes share 90–98% homology with enJSRV in deduced amino-acid sequence[1, 2, 17, 21]. The high level of homology makes it impossible to detect any circulating antibody in peripheral blood[12, 24]. One possible explanation for this phenomena is that the existence of enJSRV during the embryonic development periods makes the host tolerant towards exJSRV infection. However, the tolerance mechanisms of the disease are poorly understood, and the research on OPA moves forward slowly.
OPA represents a good model for studying the interactions between endogenous retroviruses and their exogenous counterparts. It is also valuable for research into human bronchioloalveolar cancer (BAC) since many clinical, radiological and histopathological features are similar in both diseases[10].
In order to analyze the establishment of immuno logical tolerance during the embryo period, we evaluated the expression of the enJSRVs envelope (env) protein and Hyal-2 mRNA in the immune organs of fetuses and lambs by In Situ Hybridization and Real-Time reverse transcription PCR methods. The data obtained in the present study provide useful information for the understanding of the immune tolerance pathogenesis.
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For the experiments, the Animal Center of Inner Mongolia Agricultural University offered 18 Mongolia ewes which were bred under the same nutritional condition, were oestrus at a scheduled time and became pregnant. Fetuses on day 70 of gestation, fetuses on day 130 of gestation and 3 day old newborn lambs were assigned randomly to be necropsied (n=6 ovines/period). The lungs, thymus, spleen and mesenteric lymph nodes were collected at postmortem examination, snap frozen in liquid nitrogen and stored at -80 ℃ for RNA extraction. Total RNA was isolated from tissues according to the manufacturer's recommendations (RNA extraction kit, TaKaRa, Dalian, China). The integrity of RNA was examined by gel electrophoresis in a denaturing 1.0% agarose gel and the RNA was quantified at 260 nm in a micro-spectrophotometer at an OD 260/280 ratio of 1.8 to 2.0 for all samples, and then stored at -80 ℃. All tissues were also fixed in fresh 4% paraformaldehyde for 10 h, dehydrated through a graded series of alcohol to xylene, embedded in paraffin and cut at 5 μm for In Situ Hybridization.
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Primers and probes used were designed on the basis of published enJSRV, Hyal-2, β-actins′ sequences and synthesized by TaKaRa Biotechnology Co., Ltd. (TaKaRa, Dalian, China). The information of objective genes is shown in Table 1.
Table 1. Primers and probes used in this study
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Synthesis of DNA was carried out in 50 μL reaction volumes as follows. 0.5 μL PrimeScript RTase(for 1 Step), 1 μL RNase Inhibitor (40 U/μL), 1 μL TaKaRa Ex Taq HS(5 U/μL), 2 μL dNTP Mixture(10 mmol/L each), 5 μL 10×One Step RT-PCR Buffer, 1 μL One Step Enhancer Solution, 1 μL Forward Primer(20 mmol/L), 1 μL Reverse Primer(20 mmol/L), Total RNA, and RNase free dH2O. The Conditions were 50 ℃ for 30 min, 94 ℃ for 2 min, and 45 cycles of 94 ℃ for 30 s, 30 s annealing (enJSRV 55 ℃, Hyal-2 60 ℃, and β-actin 58 ℃) and with a final extension of 72 ℃ for 1 min. RNase free dH2O in RT-PCR kits were used instead of template RNA as control. The PCR products were purified by Gel Extraction Kit (TaKaRa, Dalian, China) according to the manufacturers' instructions.
A portion of the purified PCR products was separated in a 1.5% agarose gel and visualized by ethidium bromide staining using an imaging system. The density and A260/A280 were analyzed with a micro ultraviolet spectrophotometer to identify the relative expression quantity of the target gene. Total RNA of all samples were used in RT-PCR experiments. The RT-PCR products were sequenced and analyzed with NCBI Blast. At the same time, two sense probes were synthesized according to the sequences of enJSRV and Hyal-2. Then the RT-PCR products of thymus of 70 d fetuses were used to prepare anti sense probes.
The procedure was performed as follows: 1 μg purified template DNA and RNase free dH2O were added to a final volume of 16 μL to a reaction vial, the DNA was denatured by heating it in a boiling water bath for 10 min and then quickly cooled in an ice/water bath. 4 μL DIG-High Prime was added to the denatured DNA, then mixed by centrifuging briefly. The mixture was incubated for 20 h at 37 ℃, then 2 μL 0.2 mol/L EDTA (pH 8.0) was added to stop the reaction. 1 μL labeled probes and labeled control DNA were applied to the nylon membrane. The nucleic acid was fixed to the membrane by baking for 30 min at 120 ℃. The membrane was then transferred into a plastic container with 20 mL maleic acid buffer, and incubated by shaking for 2 min at 20 ℃, incubated for 30 min in 10 mL blocking solution, incubated for 30 min in 10 mL antibody solution, washed with 10 mL washing buffer for 2×15 min, equilibrated 5 min in 10 mL detection buffer, incubated in 2 mL freshly prepared color substrate solution in a appropriate container in the dark. The reaction was stopped by washing the membrane for 5 min with 50 mL RNase free dH2O. Results were documented by digital photography.
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The procedure was performed as follows: The slide (5 μm) was dewaxed through xylene, dehydrated and digested with protease K/0.1 mol/L PBS (pH 7.4) for 1-2 min. Glycine was added to inhibit digestion, the slide was washed twice with PBS and 4% paraformaldehyde/0.1 mol/L PBS respectively. 20 μL pre-hybridization mixture (5×SSC, 5×Denhardt, 50% Deionized formamide, 1% SDS, 200 μg/mL Salmon sperm DNA) was added and pre-hybrided for 1 h at 42 ℃. The pre-hybridization mixture was removed and replaced with hybridization mixture (5×SSC, 5×Denhardt, 50% Deionized formamide, 1% SDS, 250 μg/mL Salmon sperm DNA, 10% Dextran sulfate, probe)for 18 h at 42 ℃. The sample was then washed as follows: SSC, closed fluid, anti-digoxigenin AP fluid, malefic acid buffer solution, colored buffer solution, NBT/BCIP colored solution, RNase free dH2O, and counterstained with eosin, dehydrated through a graded series of alcohol to xylene and protected with a cover slip. In the negative control, the sections were analyzed by In situ hybridization with DIG-labeled sense probes.
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First strand cDNA synthesis was performed according to the protocol suggested for the Reverse Transcription System (TaKaRa, Dalian, China) using Oligo(dT)16 primers. Quantification of mRNA levels of the genes was achieved using a Premix Ex TaqTMkit (TaKaRa, Dalian, China) using β-actin as the housekeeping gene. PCR amplification was run using IQTM5 Multicolor Real-Time PCR Detection System (BIO-RAD, America) and carried out in 25 μL reaction volume containing 2 μL cDNA, 12.5 μL SYBR Premix Ex Taq (TaKaRa, Dalian, China), 0.5 μL Forward primer (2 μm), 0.5 μL Reverse primer(2 μm), 2 μL TaqMan probes (0.2 μm) and 8.5 μL RNase free dH2O to make a total volume of 25 μL. The primers and probes used were synthesized by TaKaRa Biotechnology Co., Ltd. (TaKaRa, Dalian, China) and are shown in Table.1. All PCR reactions were run in duplicate and were performed with 45 cycles, including a negative control consisting of PCR-grade water. Data analysis was performed using a relative standard curve and based on the ratio of the fluorescent change observed with the target gene to the fluorescent change observed with β-actin. The expression level was analyzed by one-way ANOVA followed by Duncan's test. The values were expressed as mean ± SD (n = 6).