Construction and identification of recombinant lentivirus expressing small interfering RNA against human telomerase reverse transcriptase gene

doi:10.3969/j.issn.2095-4344.2014.11.014

Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (11): 1724-1729.doi: 10.3969/j.issn.2095-4344.2014.11.014

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Construction and identification of recombinant lentivirus expressing small interfering RNA against human telomerase reverse transcriptase gene

Song Yang¹, Xu Tao¹, Yang Ming-kun², Wang Guo-qi¹, Zhang En-feng¹, Sheng Wei-bin¹

¹Department of Spine Surgery, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China; ²Department of Orthopedics, Bazhong Central Hospital, Bazhong 636600, Sichuan Province, China

Revised:2014-02-25 Online:2014-03-12 Published:2014-03-12
Contact: Sheng Wei-bin, M.D., Professor, Chief physician, Department of Spine Surgery, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China
About author:Song Yang, Studying for master’s degree, Department of Spine Surgery, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China
Supported by:
the National Natural Science Foundation of China, No. 81060106

Abstract

Abstract:

BACKGROUND: Telomerase reverse transcriptase (TERT) plays an important role in telomerase activation, however there is rare report addressing the construction of the lentivirus targeted its genes to inhibit its expression in the spinal cord astrocytes.

OBJECTIVE: To construct recombinant lentivirus vector expressing small interfering RNA against TERT gene and to evaluate its potential for inhibiting the TERT expression.

METHODS: After shRNA-TERT sequence was designed and synthesized, the sequence was amplified by PCR and then connected to plasmid pLentilox3.7U6-hTERT to construct recombinant plasmid. The recombinant plasmid was then transfected to DH5α cells to screen positive colony, and the sequence was identified. The recombinant plasmid pLentilox3.7U6-TERT was transfected in 293T cells, generating recombinant lentivirus Le-TERT. The titer of recombinant lentivirus was determined and Le-TERT was transfected into the rat spinal cord astrocytes. The expression of TERT in astrocytes was detected by RT-PCR, western blot and immunofluorescence assay.

RESULTS AND CONCLUSION: The gene sequencing analysis confirmed that, recombinant plasmid pLentilox3.7U6-TERT was successfully constructed. The real-time quantitative PCR, western blot analysis and immunofluorescence assay indicated that, after Le-TERT was transfected in the astrocytes for 4 days, the inhibition rate of TERT mRNA was (63.98±2.6)%, and Le-TERT was lowly expressed in the transfected astrocytes. Recombinant expression vector pLentilox3.7U6-TERT can produce the lentivirus at high titer and effectively inhibit TERT expression in the transfected astrocytes.

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, plasmid

CLC Number:

R394.2

Song Yang, Xu Tao, Yang Ming-kun, Wang Guo-qi, Zhang En-feng, Sheng Wei-bin. Construction and identification of recombinant lentivirus expressing small interfering RNA against human telomerase reverse transcriptase gene[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(11): 1724-1729.

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Construction and identification of recombinant lentivirus expressing small interfering RNA against human telomerase reverse transcriptase gene

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