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RISS 인기검색어
- 검색키워드
- 저자 : Deshun Shi (검색결과 3 건)
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Zhaocheng Xiong,Haihang Zhang,Ben Huang,Qingyou Liu,Yingqun Wang,Deshun Shi,Xiangping Li 아세아·태평양축산학회 2018 Animal Bioscience Vol.31 No.11
Objective: The aim of the current study is to investigate the relationship between prohibitin (PHB), capping actin protein of muscle Z-line beta subunit (CAPZB), and tektin-2 (TEKT2) and sperm motility in Murrah buffalo. Methods: We collected the high-motility and low-motility semen samples, testis, ovary, muscle, kidney, liver, brain and pituitary from Murrah buffalo, and analysed the expression of PHB, CAPZB, and TEKT2 in mRNA (message RNA) and protein level. Results: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) result showed that the expression of PHB was higher and CAPZB, TEKT2 were specifically expressed in testis as compared to the other 6 tissues, and that in testis, the expression of TEKT2 was higher than that of CAPZB and PHB. Immunohistochemistry test revealed that all three genes were located on the convoluted seminiferous tubule and enriched in spermatogenic cells. Both qRT-PCR and Western Blot results showed that the expression levels of PHB, CAPZB, and TEKT2 were significantly lower in the low-motility semen group compared to the high-motility semen group (p<0.05). Conclusion: The expression levels of PHB, CAPZB, and TEKT2 in Murrah buffalo sperm have a high positive correlation with sperm motility. And the three genes may be potential molecular markers for the decline of buffalo sperm motility.
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An Improved Tet-on System to Tightly Conditionally Regulate Reporter Gene Expression
Yanping Ren,Xiangping Li,Qingyou Liu,Yanfei Deng,Deshun Shi 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.1
Reporter genes are often used as markers totrack the integration and expression of target genes inanimal genetic engineering. To avoid potential side effectsfrom reporter genes, in this study an improved Tet-onsystem was developed to control reporter gene expression,and its effectiveness was explored in transgenic cells. First,the rtTA protein was fused with Tat and NLS proteins toobtain the prokaryotic expression vector pET32a-Tat-rtTANSL. A eukaryotic transgenic vector was constructed, p-HS4-BPA-TmA-HS4, in which the reporter (mCherry) andtarget (PRL) genes were promoted by TRE and BCN,respectively. After confirming the functionality of thetransgenic vector, purified rtTA protein and Dox wereadded to induce expression of the mCherry gene. Theoptimal amount of purified rtTA protein, its influence ontarget gene expression, and the time of rtTA protein actionwere each investigated separately. The results showed thatrtTA protein was expressed in transformed E. coli with IPTGinduction. TRE could promote mCherry gene expressionby co-transfecting the constructed transgenic vector andprtTA plasmid. When purified rtTA protein and Dox wereadded, red fluorescence was observed in Bcap-37 cellstransfected with the p-HS4-BPA-TmA-HS4 vector, and theexogenous PRL gene was expressed regardless of mCherrygene expression. The optimal amount of rtTA protein was16 μg/mL, and it needed about 6 h to promote mCherrygene expression in transfected cells. These resultsdemonstrate that the expression of the mCherry reportergene can be tightly and conditionally regulated by our Tetonsystem.
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Isolation and Identification of Prepubertal Buffalo (Bubalus bubalis) Spermatogonial Stem Cells
Feng, Wanyou,Chen, Shibei,Do, Dagiang,Liu, Qinyou,Deng, Yanfei,Lei, Xiaocan,Luo, Chan,Huang, Ben,Shi, Deshun Asian Australasian Association of Animal Productio 2016 Animal Bioscience Vol.29 No.10
Isolation and culture of spermatogonial stem cells (SSCs) are attractive for production of genetic modified offspring. In the present study, buffalo spermatogonial stem-like cells were isolated, cultured and expression pattern of different germ cell marker genes were determined. To recover spermatogonia, testes from age 3 to 7 months of buffalo were decapsulated, and seminiferous tubules were enzymatically dissociated. Two types of cells, immature sertoli cell and type A spermatogonia were observed in buffalo testes in this stage. Germ cell marker genes, OCT3/4 (Pou5f1), THY-1, c-kit, PGP9.5 (UCHL-1) and Dolichos biflorus agglutinin, were determined to be expressed both in mRNA and protein level by reverse transcription polymerase chain reaction and immunostaining in buffalo testes and buffalo spermatogonial stem-like cells, respectively. In the following, when the isolated buffalo buffalo spermatogonial stem-like cells were cultured in the medium supplemented 2.5% fetal bovine serum and 40 ng/mL glial cell-derived neurotrophic factor medium, SSCs proliferation efficiency and colony number were significantly improved than those of other groups (p<0.05). These findings may help in isolation and establishing long term in vitro culture system for buffalo spermatogonial stem-like cells, and accelerating the generation of genetic modified buffaloes.
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