| Metadata |
| datasetIdentifier | PASS00797 |
| datasetType | MSMS |
| submitter | Hegang Li <lihegang2000@aliyun.com> |
| submitter_organization | Qingdao Institute of Animal Science and Veterinary Medicine |
| lab_head_full_name | Hegang Li |
| lab_head_email | lihegang2000@aliyun.com |
| lab_head_organization | Qingdao Institute of Animal Science and Veterinary Medicine |
| lab_head_country | China |
| datasetTag | Sheepskinfollicle |
| datasetTitle | Discovery of genes and proteins regulating wool diameter |
| publicReleaseDate | 2020-12-31 00:00:00 |
| finalizedDate | 2016-01-14 00:45:58 |
| summary | Sheep provide considerable materials for the animal fibre industry. Identifying genes of major effect for wool growth would offer strategies for improving the quality of fine wool. In this study, we employed Agilent Sheep Gene Expression Microarray and proteomic technology to investigate the gene expression patterns of body side skin (more wool growing) in Aohan fine wool sheep (a Chinese indigenous breed) in comparison with groin skin (no wool growing) at the anagen stage of wool follicle. Microarray analysis revealed that the majority of the genes possibly related to the wool growth could be assigned into the categories including regulation of cell division, intermediate filament, cytoskeletal part and growth factor activity. Several potential gene families might participate in hair growth regulation, including fibroblast growth factors, transforming growth factor-β, WNTs, insulin-like growth factor, vascular endothelial growth factors and so on. Proteomic analysis also indentified hundreds of differentially expressed proteins. Furthermore, according to the results at both mRNA and protein levels, similar regulation mechanism of gene activity might be engaged during skin development and embryo development. |
| contributors | Hegang Li |
| publication | unpublished |
| growth | These six sheep were reared normally in pens. |
| treatment | All animals were treated in accordance with the animal protocols defined by national and local animal welfare bodies, and all animal work was approved by the Shandong Province Biological Studies Animal Care and Use Committee.One ram and two ewes of 12-month-old Aohan fine wool sheep were used in the microarray study. These animals were half sibs (sharing the same father). In August 2010, two areas of full-thickness skin were sampled from the same animal under local anaesthesia: body side skin (wool bearing) and groin skin (non-wool bearing) for microarray and proteomic experiments. The area of each sample was about 1 cm2. |
| extraction | Lysis buffer preparation: 42% Urea, 15.2% Thiourea, 4% CHAPS, 1% DTT. Sampled tissues were homogenziated in lysis buffer (containing 1% cocktail and 2% IPG-buffer, added right before use) at the ratio of 1:7 (weight/volume). The tissues were cut into small pieces by ophthalmic scissors, and left at 4℃ for 1h, vortexed it every 15 min. Then, the tissue homogenate was centrifuge at 40,000g for 30 min. Supernatants were collected and stored at -80℃. Protein concerntrations were determined by Bradford method. |
| separation | 1.One-dimensional electrophoresis
0.5% IPG-buffer was added into each 150 μg protein sample (in a final volume of 400-600μL), and was loaded in the One-dimensional electrophoresis instrument. The progamme is as follows: Step-n-hold (S1, 30V for 6h; S2, 60V for 6h); Gradient (S3, 500V for 1h;S4,1000V for 1h; S5, 3000V for 3h; S6, 8000V for 3h); Step-n-hold (S7, 8000V for 20h).
2.2-dimensional (2-D) SDS-PAGE preparation
Tris-HCl (PH=8.8), Monomer storage (30% Acrylamid and 0.8% NN’-methy lenebisacry lamid), 10×electrophoresis buffer (3.03% Tris-Base, 14.4% Glycine, 1% SDS), balanced solution (36.05% Urea, 5% Tris-HCl, 2% SDS, 34.5% Glycerine).
3.The second dimensional SDS-PAGE
The electrophoresis programme is as follows:
Transfer: Voltage 300v, Current 50mA, Time 1h.
Separation: Voltage 300v, Current 200~250mA, Time 4~5h.
Fixative preparation: 40% Ethanol and 10% Acetic acid.
Electrophoresis was carried out until the blue dye front had just disappeared from the bottom of the gel.
Fixation: take out the rubber strip and put it into Fixative for 1h.
4.Staining and visualization
Sensitizing solution preparation: 30% Ethanol, 0.314% Na2S2O3, 6.8%NaAc.
Sensitizing: the gels were sensitized for 30 min.
Washing: the gels were washed for three times using ddH2O. Ten minutes each time.
Ilver staining: Silver staining solution was prepared (1.25 AgNO3 and 200μL Formaldehyde in 500mL ddH2O). The gels were stained by the solution for 20 min.
Washing: the gels were washed for 2 min using ddH2O.
Visualizing solution preparation: 12.5g Na2CO3 and 100μL Formaldehyde in 500mL ddH2O.
Termination solution preparation: 2g Glycine in 50ml ddH2O.
Visualization until the solution became muddy, then terminating for 30min. |
| digestion | |
| acquisition | |
| informatics | Determination of relative protein expression
Gels were then scanned and analyzed using ImageMaster TM 2D platinum software (Version 5.0, GE Healthcare, San Francisco, CA, USA). The expression level was determined by the relative volume of each spot in the gel and expressed as %Vol (%Vol = [spot volume / Σvolumes of all spots resolved in the gel]). The means and standard deviations of both sample groups were calculated. Statistical significance with Student’s t-tests using ImageMaster TM 2D platinum software. P values﹤0.05 were considered statistically significant. |
| instruments | Ultraflex II MALDI-TOF-TOF mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) |
| species | Sheep |
| massModifications | none |
