Methodology on construction of rat aldehyde dehydrogenase 2 gene regulation recombinant adenovirus vectors
10.13699/j.cnki.1001-6821.2014.10.016
- VernacularTitle:大鼠乙醛脱氢酶2基因调控腺病毒载体构建方法及意义
- Author:
Hong-Bo LI
1
;
Xiao-E LANG
Author Information
1. 北京协和医学院临床医学 系
- Keywords:
aldehyde dehydrogenase 2;
adenovirus vector;
ischemia /reperfusion;
cardioprotection
- From:
The Chinese Journal of Clinical Pharmacology
2014;(10):922-925
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct adenovirus specific for rat aldehyde dehydrogenase 2 (ALDH2) gene interference and consistent activation and transfect the viruses into rat cardiomyocytes to observe transfection effect and its influence on ALDH2 expression.Methods Consistently active ALDH2 (CA -ALDH2) mutant gene was amplified and linked to shuttle vector, thus recombinant shuttle plasmid was subsequently con -structed.Stem -loop DNA for ALDH2 silencing RNA ( ALDH2 -siRNA) sequence was synthesized and loaded it into vector thus recombi -nant shuttle plasmid was constructed .Both kinds of plasmid were imple -mented amplification and enzyme identification.Verified plasmids were loaded into pAdeno adenovirus vectors.The viruses were then transfected into 293 cell linage to replicate and be purified.Treat cultured cardio-myocytes from 1 -day -old neonatal male Sprague Dawley (SD) rat with empty adenovirus vector control and both kinds of recombinant adenovirus vector, and perform subsequent assay for ALDH2 expression.Results Both vectors are identified by endonuclease with titre of 2 ×10 10 , 1.6 ×10 10 PFU ? mL-1 respectively after purification.The change in ALDH2 expression after infection are both observable ( P <0.01 ) . Conclusion Both of the double -way regulation recombinant vectors for rat ALDH2 gene are successfully constructed , which are capable of effec-tive regulation of the gene expression in rat cardiomyocytes .
