中国药理学与毒理学杂志2013，Vol.27Issue(1)：29-33,5.DOI:10.3867/j.issn.1000-3002.2013.01.006
ErbB4基因miRNA慢病毒载体的构建及其在小鼠海马齿状回的表达
Construction of lentiviral miRNA vector targeting ErbB4 gene and its expression in dentate gyrus of mouse hippocampus
摘要
Abstract
OBJECTIVE To construct a lentiviral vector containing miRNA targeting ErbB4, package high titer lentivirus and observe its infection and expression in the hippocampus dentate gyms of mice. METHODS Lentiviral miRNA vector targeting ErbB4 was constructed from miRNA expression vectors by Gateway recombinant technology. HEK293T producer cell line was contransfected with the lentiviral expression construct and packaging mix, viral supernatant was harvested and condensed, and the titer was determined using green fluorescent protein (GFP) expression with flow cytometry. Lentivirus was stereotactically micro-infused into the hippocampus dentate gyrus of mice. Six months later, brain cryosections were used to observe gene expression. Immunofluorescence staining of brain sections with antibodies specific for neurons marker neuronal nuclei (NeuN) and astrocytes marker glial fibrillary acidic protein (GFAP) was used to analyze lentivirus transduced cell types. RESULTS Lentiviral miRNA vector targeting ErbB4 was validated by sequencing, showing the correct construction. Lentivirus were harvest and concentrated after packaging in HEK293T cells. After transduction HEK293T cells with 10 -fold serial dilutions of lentivirus stocks, GFP positive cells were detected by flow cytometry and the lentiviral titer was determined to be 1.0 ×1012 transduction units (TU) ·L-1. Lentivirus mediated expression using GFP as a reporter were observed on hippocampus dentate gyrus in mouse brain sections 6 months after stereotactic micro-infusion lentivirus 1.0 ×1012 TU·L-1. In immunofluorescent staining analysis, most GFP positive cells were labelled for NeuN and no cell was double-labelled for GFP and GFAP. CONCLUSION ErbB4 specific lentiviral miRNA vector is successfully constructed. High titer lentiviral stocks are prepared and transduced brain tissue in vivo successfully and stably. Neurons are the primary cell type transduced by lentivirus.关键词
慢病毒/ErbB4/海马/齿状回Key words
lentivirus/ ErbB4/ hippocampus/ dentate gyrus分类
医药卫生引用本文复制引用
李树玲,颜慧,宫泽辉..ErbB4基因miRNA慢病毒载体的构建及其在小鼠海马齿状回的表达[J].中国药理学与毒理学杂志,2013,27(1):29-33,5.基金项目
国家十一五科技重大专项综合性新药研究开发技术平台(2009ZX09301-002) （2009ZX09301-002）
