Construction of lentiviral vector containing sirt1 gene and its expression in retinal ganglion cell_Chinese Journal of Ocular Fundus Diseases

Authors：

YaoQing , ZhangJirong , YangYi , ZhangQian , LiJianning ,  SunYuning

Department of Biochemistry and Molecular Biology, School of Basic Medicine, Ningxia medical university, Yinchuan 750004, China;

Corresponding author：

SunYuning, Email: 329545694@qq.com

Keywords：

Silent information regulator proteins, saccharomyces cerevisiae; Retinal ganglion cells; Lentivirus infections; Cells, cultured

DOI：

10.3760/cma.j.issn.1005-1015.2016.01.016

Video：

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Abstract Full text Figures/Tables Video References Cited by

Objective To construct a lentiviral vector carrying rat sirt1 gene and observe the expression of sirt1 in retinal ganglion cell (RGC) of rat. Methods Rat sirt1 cDNA was inserted into pLV5 vector. After identification by sequencing analysis and PCR, the recombinant sirt1expressinglentivirus vector was packaged by cotransfecting 293T cells with packaged plasmid.Then pLV5-sirt1 was used to infect the cultured Sprague-Dawley rat RGC cell in vitro.The expressions of sirt1 protein and mRNA in infected rat RGC were detected by quantitative real-time PCR and Western blot. Results The sirt1 expression vector pLV5 was successful constructed and sequence was proved to be correct. The expression of sirt1 protein and mRNA in RGC was significantly increased than that in cells infected with control lentiviruses(P < 0.05). Conclusion We have successful constructed a sirt1 expression lentivirus vector pLV5-sirt1 and it can increase the expression of sirt1 protein and mRNA in the rat retinal ganglion cells.

Citation： YaoQing, ZhangJirong, YangYi, ZhangQian, LiJianning, SunYuning. Construction of lentiviral vector containing sirt1 gene and its expression in retinal ganglion cell. Chinese Journal of Ocular Fundus Diseases, 2016, 32(1): 66-69. doi: 10.3760/cma.j.issn.1005-1015.2016.01.016 Copy

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2.	Knight JR, Milner J. SIRT1, metabolism and cancer[J].Curr Opin Oncol, 2012, 24(1): 68-75. DOI: 10.1097/CCO.0b013e32834d813b.
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11.	Dull T, Zufferey R, Kelly M, et al. A third-generation lentivirus vector with a conditional packaging system[J].J Virol, 1998, 72(11):8463-8471.
12.	Mitta B, Rimann M, Fussenegger M. Detailed design and comparative analysis of protocols for optimized production of high-performance HIV-1-derived lentiviral particles[J]. Metab Eng, 2005, 7(5-6):426-436.
13.	郑敬民, 尹广, 赵文紧, 等. C3aR慢病毒表达载体的构建及在肾小管上皮细胞中的转染实验[J].医学研究生学报, 2015, 28(4):355-358.Zheng JM, Yin G, Zhao WJ, et al.Construction of a C3aR expression vector and infection in tubular epithelial cells[J].J Med Postgra, 2015, 28(4):355-358.
14.	吴倩, 魏亚明, 李瑜元, 等.慢病毒介导稳定过表达β-catenin可促进间充质干细胞的增殖和迁移[J].中国组织工程研究, 2013, 17(40):7076-7083. DOI:10.3969/j.issn.2095-4344.2013.40.009.Wu Q, Wei YaM, Li YY, et al. Lentivirus-mediated over-expression of beta-catenin accelerates proliferation and migration of mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(40):7076-7083. DOI:10.3969/j.issn.2095-4344.2013.40.009.

1. García-Ayuso D, Salinas-Navarro M, Nadal-Nicolás FM, et al.Sectorial loss of retinal ganglion cells in inherited photoreceptor degeneration is due to RGC death[J].Br J Ophthalmol, 2014, 98(3):396-401. DOI: 10.1136/bjophthalmol-2013-303958.
2. Knight JR, Milner J. SIRT1, metabolism and cancer[J].Curr Opin Oncol, 2012, 24(1): 68-75. DOI: 10.1097/CCO.0b013e32834d813b.
3. Ou X, Lee MR, Huang X, et al. SIRT1 positively regulates autophagy and mitochondriafunction in embryonic stem cells under oxidative stress[J].Stem Cells, 2014, 32(5):1183-1194. DOI: 10.1002/stem.1641.
4. 张燕, 李红阳, 曹咏梅.沉默信息调节因子1调控胆固醇合成对大鼠视神经损伤后RGCs修复的作用机制[J].国际眼科杂志, 2014, 14(10):1760-1764.Zhang Y, Li HY, Cao YM. Effect of SIRT1 regulating cholesterol synthesis in repairing retinal ganglion cells after optic nerve injury in rats[J]. Int Eye Sci, 2014, 14(10):1760-1764.
5. Chen W, Bhatia R.Roles of SIRT1 in leukemogenesiss[J]. Curr Opin Hematol, 2013, 20(4):308-313. DOI: 10.1097/MOH.0b013e328360ab64.
6. Kitada M, Kume S, Koya D. Role of sirtuins in kidney diseases[J]. Curr Opin Nephrol Hypertens, 2014, 23(1):75-79. DOI: 10.1097/01.mnh.0000437330.85675.ac.
7. Zhu GY, Shi BZ, Li Y. FoxM1 regulates Sirt1 expression in glioma cells[J]. Eur Rev Med Pharmacol Sci, 2014, 18(2): 205-211.
8. Mimura T, Kaji Y, Noma H, et al. The role of SIRT1 in ocular aging[J].Exp Eye Res, 2013, 116:17-26. DOI: 10.1016/j.exer.2013.07.017.
9. Chen S, Fan Q, Li A, et al.Dynamic mobilization of PGC-1αmediates mitochondrial biogenesis for the protection of RGC-5 cells by resveratrol during serum deprivation[J].Apoptosis, 2013, 18(7):786-799. DOI: 10.1007/s10495-013-0837-3.
10. 张燕, 卢博喻, 张慧敏, 等. sirt1作为靶点治疗年龄相关性视网膜病变的研究进展[J].国际眼科杂志, 2015, 15(2):233-237.Zhang Y, Lu BY, Zhang HM, et al.Research progress of SIRT1 as a target for the treatment of age-related retinal disease[J].Int Eye Sci, 2015, 15(2): 233-237.
11. Dull T, Zufferey R, Kelly M, et al. A third-generation lentivirus vector with a conditional packaging system[J].J Virol, 1998, 72(11):8463-8471.
12. Mitta B, Rimann M, Fussenegger M. Detailed design and comparative analysis of protocols for optimized production of high-performance HIV-1-derived lentiviral particles[J]. Metab Eng, 2005, 7(5-6):426-436.
13. 郑敬民, 尹广, 赵文紧, 等. C3aR慢病毒表达载体的构建及在肾小管上皮细胞中的转染实验[J].医学研究生学报, 2015, 28(4):355-358.Zheng JM, Yin G, Zhao WJ, et al.Construction of a C3aR expression vector and infection in tubular epithelial cells[J].J Med Postgra, 2015, 28(4):355-358.
14. 吴倩, 魏亚明, 李瑜元, 等.慢病毒介导稳定过表达β-catenin可促进间充质干细胞的增殖和迁移[J].中国组织工程研究, 2013, 17(40):7076-7083. DOI:10.3969/j.issn.2095-4344.2013.40.009.Wu Q, Wei YaM, Li YY, et al. Lentivirus-mediated over-expression of beta-catenin accelerates proliferation and migration of mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(40):7076-7083. DOI:10.3969/j.issn.2095-4344.2013.40.009.

Chinese Journal of Ocular Fundus Diseases

Construction of lentiviral vector containing sirt1 gene and its expression in retinal ganglion cell

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