Preparation of anti-mouse caspase-12 mRNA hammerhead ribozyme and identification of its activity in vitro.
OBJECTIVE
To prepare and identify specific anti-mouse caspase-12 hammerhead ribozymes in vitro, in order to select a more effective ribozyme against mouse caspase-12 as a potential tool to rescue cells from endoplasmic reticulum stress induced apoptosis.


METHODS
Two hammerhead ribozymes directed separately against 138 and 218 site of nucleotide of mouse caspase-12 mRNA were designed by computer software, and their DNA sequences were synthesized. The synthesized ribozymes were cloned into an eukaryotic expression vector-neo(r)pBSKU6 and embedded in U6 SnRNA context for further study. Mouse caspase-12 gene segment was cloned into PGEM-T vector under the control of T7 RNA polymerase promoter (containing gene sequence from positions nt 41 to nt 894) as target. In vitro transcription both the ribozymes and target utilize T7 promoter. The target was labeled with (alpha- (32)P)UTP, while ribozymes were not labeled. After gel purification the RNAs were dissolved in RNase free water. Ribozyme and target were incubated for 90 min at 37 degrees in reaction buffer (40 mmol/L Tris-HCL, pH 7.5, 10 mmol/L Mg(2+)). Molar ratio of ribozyme vs target was 30:1. Samples were analyzed on 6% PAGE(containing 8 mol/L urea).


RESULTS
Both caspase-12 and ribozyme gene sequences were successfully cloned into expression vector confirmed by sequencing. Ribozymes and caspase-12 mRNA were obtained by in vitro transcription. Cleavage experiment showed that in a physiological similar condition (37 degrees, pH 7.5), Rz138 and Rz218 both cleaved targets at predicted sites, for Rz138 the cleavage efficiency was about 100%, for Rz218 the value was 36.66%.


CONCLUSIONS
Rz138 prepared in vitro can site specific cleave mouse caspase-12 mRNA with an excellent efficiency. It shows a potential to suppress the expression of caspase-12 in vivo, thus provided a new way to protect cells from ER stress induced apoptosis.
Jiang
Shan
S
Department of Infectious Disease, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Xie
Qing
Q
Zhang
Wei
W
Zhou
Xia-Qiu
XQ
Jin
You-Xin
YX
D000818:Animals; D053454:Caspase 12; D020169:Caspases / Q000235:genetics*; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D007700:Kinetics; D051379:Mice; D016337:RNA, Catalytic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D018076:DNA, Complementary; D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme; C506440:Casp12 protein, mouse; D053454:Caspase 12; D020169:Caspases