Construction and identification of a specific small interfering RNA expression vector of Caspase-12 in mouse hepatoma cell line
- VernacularTitle:小鼠半胱天冬酶-12特异性小干扰RNA真核表达载体的构建及鉴定
- Author:
Lan-Yi LIN
;
Qing XIE
;
Hui WANG
;
Shan JIANG
;
Xia ZHOU
;
Liu QIU
;
Ye YUN
;
Hong YU
;
Qing GUO
;
You-Xin JIN
- Publication Type:Journal Article
- Keywords:
Endoplasmic reticulum stress;
Apoptosis;
Caspases;
RNA interference
- From:
Chinese Journal of Infectious Diseases
2000;0(02):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a specific small interfering double-stranded RNA(siRNA) expression vector of Caspase-12 and to evaluate inhibitory effect of this siRNA on caspase-12 mRNA activity.Methods Three groups of siRNA targeting different gene sites of caspase-12 were designed and synthesized chemically.Mouse hepatoma cell line,Hepa1-6,was transfected with the siRNA by 24 h.Reverse transcription-polymerase chain reaction(RT-PCR)was performed to analyze the inhibi- tion of caspase-12.Then the most effective siRNA was selected and the two template sequences for the siRNA were inserted into pRNAT-H1.1Neo expression vector.The recombinant plasmid, referred to as pRNAT-casp12,was verified by PCR analysis and sequencing.The expression of caspase-12 at mRNA and protein level,after transfection with pRNAT-casp12 by 48 h and 72 h respectively,were analyzed by using real-time PCR and Western blotting.Results The chemically synthesized siRNA*1 and siRNA*3 could inhibit mouse hepatoma cell caspase-12 mRNA by 59.9% and 39.6%(P
