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

目的构建靶向ErbB4的miRNA慢病毒载体,包装产生高滴度慢病毒并观察其感染小鼠海马齿状回基因的表达。方法将靶向ErbB4基因的miRNA干扰质粒利用Gateway重组技术构建慢病毒表达质粒,并与慢病毒包装系统共转染HEK293T包装细胞,收获上清并浓缩得到慢病毒浓缩液,通过测定绿色荧光蛋白(GFP)表达水平测定其滴度。所得慢病毒浓缩液经立体定位微量注射感染小鼠海马齿状回。观察鼠脑冰冻切片基因表达情况,并用神经元标志物神经元核抗原(NeuN)和星形胶质细胞标志物胶质纤维酸性蛋白(GFAP)特异性抗体行免疫染色分析转导的细胞类型。结果阳性克隆测序结果与设计序列一致,说明靶向ErbB4基因的miRNA慢病毒干扰载体构建成功。包装收获慢病毒。浓缩的慢病毒包装上清感染HEK293T细胞后,流式细胞术检测GFP阳性率,计算得慢病毒活性滴度为1.0×1012转导单位(TU).L-1。小鼠海马齿状回立体定位微量注射1.0×1012TU.L-1慢病毒6个月后,观察到注射部位外源基因的显著表达。免疫荧光染色显示,慢病毒转导的细胞多呈NeuN标记阳性,并与GFAP的表达不重叠。结论靶向ErbB4的miRNA慢病毒载体构建成功,获得了靶向ErbB4基因的高滴度miRNA慢病毒颗粒,并实现了脑实质的局部长期稳定转导,慢病毒转导的主要细胞类型为神经元。

OBJECTIVE To construct a lentiviral vector containing miRNA targeting ErbB4, pack- age high titer lentivirus and observe its infection and expression in the hippocampus dentate gyrus 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. Lentivir- us 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 × 10^12 transduction units （TU） · L-1. Lentivirus mediated expres- sion using GFP as a reporter were observed on hippocampus dentate gyrus in mouse brain sections 6 months after stereotactic micro-infusion lentivirus 1.0 × 10^12 TU· L-1. In immunofluorescent staining anal- ysis, 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.