Construction of an adenoviral vector containing mouse Hes1 gene and its expression in the hippocampus of adult mice
10.3760/cma.j.issn.1671-8925.2011.08.002
- VernacularTitle:重组腺病毒Ad5-mHes1的构建及其在小鼠海马组织中的表达
- Author:
Rong YAN
1
;
Jie ZHAO
;
Qi ZHANG
;
Lin ZHANG
;
Lin-Chun HUAN
;
Wang-Miao ZHAO
;
Shu-Yuan YANG
;
Jian-ning NG ZHA
;
Xin-yu NG YA
Author Information
1. 天津医科大学总医院
- Keywords:
Hes1 gene;
Recombinant adenovirus;
Plasmid;
Hippocampus;
Dentate gyrus
- From:
Chinese Journal of Neuromedicine
2011;10(8):759-763
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct an adenoviral vector containing mouse Hes1 gene, observe its expression in the hippocampus of adult mice and build a basis for further investigation of Hes1 gene in adult neurogenesis. Methods The restriction endonuclease was used to digest plasmid pEGFP-mHes1 and pDC316, and then, the products were recovered and connected by T4 DNA ligase and the shuttle plasmid pDC316-mHes1 was constructed which was identified by the method of PCR and EcoRI+HindⅢ digestion. After that, the shuttle plasmid pDC316-mHes1 was cotransfected into 293 cells with the adenovirus skeleton plasmid pBHGlox_E1,3Cre to obtain the produced replication defective recombinant adenovirus Ad5-mHes1. Then, the recombinant adenovirus could be further amplified and purified. The report recombinant adenoviruses were Ad5-EGFP containing enhanced green fluorescent protein (EGFP).Then, Ad5-mHes1 and Ad5-EGFP were stereotactic injected into the hippocampus of the adult C57BL/6 mice and their expressions in the hippocampus were detected. Western blotting was used to detect the Hes1 protein level 7 d after the injection. Fluorescent microscopy was used to observe the expression of EGFP in the hippocampus. Results The experimental results of identification by the methods of PCR and EcoRI+HindⅢ digestion were in accordance with the anticipated results, and the sequences were also the same with mHeslCDS sequences; Hes1 gene was expressed in the hippocampus of both the PBS injection group and the Ad5-mHes1 injection group 7 d after the injection, and the expression of Hes1/GAPDH in Ad5-mHes1 injection hippocampus (0.705 ±0.128) was statistically different as compared with that in PBS injection group (0.363±0.053, P＜0.05). Ad5-EGFP strongly expressed in the granular cell layer and subgranular zone (SGZ) of dentate gyrus. Conclusion The adenoviral vector of mouse Hes1 gene is successfully established and Hes1 gene is expressed in the hippocampus of C57BL/6 adult mice.
