[Development of a system for quick screening of efficient HBx-siRNA].
Zhejiang Da Xue Xue Bao Yi Xue Ban
; 33(4): 300-5, 310, 2004 07.
Article
em Zh
| MEDLINE
| ID: mdl-15269979
ABSTRACT
OBJECTIVE:
To develop a system for quick screening of efficient siRNA targeted HBx mRNA.METHODS:
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.RESULT:
(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.CONCLUSION:
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Transativadores
/
RNA Interferente Pequeno
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
/
Screening_studies
Limite:
Humans
Idioma:
Zh
Ano de publicação:
2004
Tipo de documento:
Article