Preparation and identification of hammerhead ribozyme in vitro against caspase-12 mRNA fragments / 中华肝脏病杂志
Chinese Journal of Hepatology
;
(12): 121-124, 2005.
Article
Dans Chinois
| WPRIM
| ID: wpr-233591
ABSTRACT
<p><b>OBJECTIVE</b>To design and synthesize ribozymes targeting 138 and 218 sites of the mRNA nucleotide of mouse caspase-12, a key intermedium of ER stress mediated apoptosis, and to identify their activities through in vitro transcription and cleavage.</p><p><b>METHODS</b>The mouse caspase-12 gene fragment was obtained by RT-PCR and cloned into the PGEM-T vector under the control of T7 RNA polymerase promoter. The transcription product of the target was labeled with a-32P UTP, while ribozymes were not labeled. Ribozyme and target RNA were incubated for 90 min at 37 degree C in a reaction buffer to perform the cleavage reaction.</p><p><b>RESULTS</b>It was found that under a condition of 37 degree C, pH 7.5 and with Mg2+ in a concentration of 10 mmol/L, Rz138 and Rz218 both cleaved targets at predicted sites, and the cleavage efficiency of Rz138 was 100%.</p><p><b>CONCLUSION</b>Rz138 and Rz218 prepared in vitro possess the perfect specific catalytic cleavage activity. Rz138 has excellent cleavage efficiency. It may be a promising tool to prevent ER stress induced apoptosis through catalytic cleavage of caspase-12 mRNA in vivo. It also can be used to verify whether caspase-12 is necessary in ER stress induced apoptosis.</p>
Texte intégral:
Disponible
Indice:
WPRIM (Pacifique occidental)
Sujet Principal:
ARN messager
/
Données de séquences moléculaires
/
Séquence nucléotidique
/
Chimie
/
ARN catalytique
/
Stress oxydatif
/
Réticulum endoplasmique
/
Caspase-12
/
Génétique
/
Métabolisme
Limites du sujet:
Animaux
langue:
Chinois
Texte intégral:
Chinese Journal of Hepatology
Année:
2005
Type:
Article
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