Construction and identification of recombinant lentivirus expressing small interfering RNA against human telomerase reverse transcriptase gene / 中国组织工程研究

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 smal 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 DH5αcel s to screen positive colony, and the sequence was identified. The recombinant plasmid pLentilox3.7U6-TERT was transfected in 293T cel s, 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 successful y 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.