Selecting functional siRNA target sites of hUBE2W based on H1-U6 dual promoter RNAi plasmid / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 1975-1980, 2008.
Article
Dans Chinois
| WPRIM
| ID: wpr-302882
ABSTRACT
hUBEW, a newly identified class I ubiquitin conjugating enzyme, probably plays an important role in tumorigenesis and DNA repair processes. RNA interference (RNAi) is a process in cells to degrade specific homologous mRNA by forming duplex RNA and has been developed into a powerful tool to study gene functions. In this study, the H1-U6 dual promoter RNAi plasmid was constructed and the target sequence for hUbe2w could be transcribed from both strands and form a double stranded RNA with two 5'Uridine overhangs, which closely resembles endogenous functional siRNA. The hUbe2w cDNA was amplified from reverse transcription of the 293FT total RNA by RT-PCR, and then cloned into the pGL3-Control, pCMV-myc and pDsRed-express-C1 plasmids respectively, which were selected as report vectors to detect the RNAi effects. The plasmids were co-transfected into HEK293FT cells, and then the luciferase activity and hUBE2W protein expression were measured respectively. The Resulted reduction of mRNA and protein level demonstrate that the targets of 125 and 259 could significantly inhibit the hUbe2w expression.
Texte intégral:
Disponible
Indice:
WPRIM (Pacifique occidental)
Sujet Principal:
Physiologie
/
Plasmides
/
ARN messager
/
Données de séquences moléculaires
/
Séquence nucléotidique
/
Petit ARN nucléaire
/
Régions promotrices (génétique)
/
Petit ARN interférent
/
Interférence par ARN
/
Ubiquitin-conjugating enzymes
Limites du sujet:
Humains
langue:
Chinois
Texte intégral:
Chinese Journal of Biotechnology
Année:
2008
Type:
Article
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