[Construction of a retroviral vector for RNA interference targeting human telomerase reverse transcriptase].
OBJECTIVE
To construct a recombinant retroviral vector for RNA interference targeting human telomerase reverse transcriptase (hTERT).


METHODS
The sequences coding for enhanced fluorescence protein (EGFP), U6 promoter and a small interfering RNA (siRNA) targeting hTERT were amplified by PCR, respectively, and sub-cloned sequentially into the retroviral shuttle plasmid pLXSN to construct the plasmid pLXSN-EGFP-U6-siTERT. The recombinant expression plasmid was identified by restriction enzyme digestion and sequencing. Fluorescence microscopy and flow cytometry were employed to analyze EGFP expression in NIH3T3 transfected with the recombinant plasmid, and MMT assay was performed to evaluate the growth inhibition of Hela cells resulting from RNA interference mediated by the plasmid.


RESULTS
Sequence analysis and restriction enzyme digestion showed that the recombinant expression plasmid pLXSN-EGFP-U6-siTERT was constructed successfully. Twenty-four hours after transfection of NIH3T3 cells with the recombinant plasmid, the expression rate of EGFP reached 24.1% as shown by flow cytometry. MTT assay demonstrated a cell death rate of 53.2% 72 h after transfection of Hela cells with the plasmid.


CONCLUSIONS
The successful construction of the recombinant retroviral plasmid mediating potent cell growth inhibition suggests the great potential of RNA interference technique in suppressing hTERT expression in mammalian tumor cells.
Wang
Quan-shi
QS
PET Center, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. wqslph@163.net
Wang
Xin-lu
XL
Li
Hua
H
Wang
Qiao-yu
QY
Wang
Quan-ying
QY
Yang
Guang-xiao
GX
D000818:Animals; D003001:Cloning, Molecular; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D019098:Telomerase / Q000096:biosynthesis / Q000235:genetics*
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C509186:TERT protein, human; D019098:Telomerase