Development of a system for quick screening of efficient HBx-siRNA / 浙江大学学报·医学版
Journal of Zhejiang University. Medical sciences
;
(6): 300-310, 2004.
Article
in Chinese
| WPRIM
| ID: wpr-341884
ABSTRACT
<p><b>OBJECTIVE</b>To develop a system for quick screening of efficient siRNA targeted HBx mRNA.</p><p><b>METHODS</b>Using recombination DNA technique, the fusion expression plasmid of HBx and EGFP was constructed, and siRNA expression cassettes (SECs) containing U6+1, H1 or tRNA(Val )promoter were prepared via one-step overlapping extension PCR. By co-transfection with recombinant plasmid and SECs into AD293 cell, the inhibition effects on the transient expression of HBx-EGFP fusion protein were analyzed by FACS and semi-quantitated RT-PCR analysis.</p><p><b>RESULT</b>(1)HBx-EGFP fusion protein expression plasmid pHBx-EGFP was constructed successfully, which expressed green fluorescence in cell mainly located at plasma or the periphery of nucleus in granules. (2) Co-transfection with recombinant plasmid and SECs into AD293 cells resulted in inhibition of HBx-EGFP expression. SEC-siHBx388 showed significant inhibition effect on HBx-EGFP expression compared with SEC-siHBx271, indicating that siHBx388 is effective siRNA site and could be screened out with our screening system. In addition,the results of that U6+1-, tRNA(Val) and H1-siHBx388 reduced HBx-EGFP expression by 21.7%, 12.9% and 12.4% of control respectively indicated that both tRNAVal and H1 promoter was high efficient in driving effect of siHBx388.</p><p><b>CONCLUSION</b>Combination of the HBx expression carrying reporter gene and PCR-based multi promoter SECs may develop a useful system to be applied in identification of optimal HBx- siRNA and its matching promoter.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Plasmids
/
Recombinant Fusion Proteins
/
Molecular Sequence Data
/
Base Sequence
/
Transfection
/
Genetic Therapy
/
Trans-Activators
/
Cells, Cultured
/
RNA, Small Interfering
/
Green Fluorescent Proteins
Type of study:
Diagnostic study
/
Prognostic study
/
Screening study
Limits:
Humans
Language:
Chinese
Journal:
Journal of Zhejiang University. Medical sciences
Year:
2004
Type:
Article
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