Construction of targeting short hairpin RNA plasmid vector expressing TERT gene

doi:10.3969/j.issn.2095-4344.2014.07.013

Abstract

Abstract:

BACKGROUND: Telomerase reverse transcriptase (TERT) plays an important role in telomerase activation.
OBJECTIVE: To construct the targeting short hairpin RNA plasmid vector expressing TERT gene from astrocytes by using pLentilox3.7.U6.
METHODS: By using two sequences from TERT gene, we synthetized sense and antisense strand template sequences of RNA interference molecular in vitro, and then obtained the complementary strands through annealing procedure. We connected the strands with pLentilox3.7.U6 that was sequenced and transfected into the Escherichia coli. In the end, we tested its effect of reducing the TERT gene expressing by using cultured astrocytes from rat spinal cord in vitro through western blot and immunofluorescence technique.
RESULTS AND CONCLUSION: Western blot and immunofluorescence assay showed that, compared with the control group, the interference groups had a lower TERT expression in astrocytes. The targeting short hairpin RNA plasmid vector expressing TERT gene is useful to reduce the TERT gene expression. The targeting short hairpin RNA plasmid vector expressing TERT gene is valid for us to do the further test learning the mechanism of astrocytes in spinal cord injury.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: transfection, gene expression, cells, plasmids, spinal cord injuries

CLC Number:

R318

Song Yang, Xu Tao, Yang Ming-kun, Sheng Wei-bin. Construction of targeting short hairpin RNA plasmid vector expressing TERT gene[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(7): 1057-1062.

Figures/Tables 2

References

[1] Kirkpatrick KL, Mokbel K. The significance of human telomerase reverse transcriptase (hTERT) in cancer. Eur J Surg Oncol. 2001;27(8):754-760.

[2] Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in tetrahymena extracts. Cell. 1985;43(2 Pt 1):405-413.

[3] The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.

[4] 杨镭镭,王清秀,彭成为,等.大鼠脊髓星形胶质细胞的分离与纯化[J].解剖学杂志,2008,31(2):207-209.

[5] 宋晓斌,杨智勇,邓兴力,等.星形胶质细胞瘤干细胞的体外分离, 培养与鉴定[J].中国组织工程研究,2013,17(1):56-61.

[6] Hobohm C, Gunther A, Grosche J, et al. Decomposition and long-lasting downregulation of extracellular matrix in perineuronal nets induced by focal cerebral ischemia in rats. J Neurosci Res. 2005;80(4):539-548.

[7] Alonso G. NG2 proteoglycan-expressing cells of the adult rat brain: possible involvement in the formation of glial scar astrocytes following stab wound. Glia. 2005;49(3):318-338.

[8] Kozuka N, Itofusa R, Kudo Y, et al. Lipopolysaccharide and proinflammatory cytokines require different astrocyte states to induce nitric oxide production. J Neurosci Res. 2005;82(5): 717-728.

[9] 周建军,吴国材,胡荣,等.大鼠实验性脊髓损伤后胶质瘢痕分布规律研究[J].中华实验外科杂志,2007,23:347-350.

[10] Tao X, Ming-kun Y, Wei-bin S, et al. Role of telomerase reverse transcriptase in glial scar formation after spinal cord injury in rats. Neurochem Res. 2013;38(9):1914-1920.

[11] Yu J Y, DeRuiter S L, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci U S A. 2002;99(9):6047- 6052.

[12] 李正伟,白云深,任宪盛,等.靶向RhoA基因的shRNA质粒表达载体的构建与鉴定[J].中国老年学杂志,2009,29(2):164-166.

[13] 赵鹏,傅震,尤永平,等.靶向人端粒酶反转录酶基因小片段干涉 RNA慢病毒表达载体的构建与鉴定[J].中华医学遗传学杂志, 2008,25(1):27-31.

[14] 谭挺,胡志奇.pLEGFP-N1-端粒酶反转录酶载体的构建及其表达[J].中华实验外科杂志,2011,28(12):2108-2110.

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