• 医学文献
  • 知识库
  • 评价分析
  • 全部
  • 中外期刊
  • 学位
  • 会议
  • 专利
  • 成果
  • 标准
  • 法规
  • 临床诊疗知识库
  • 中医药知识库
  • 机构
  • 作者
热搜词:
换一批
论文 期刊
高级检索

检索历史 清除

携带靶向干扰小鼠早期生长反应基因1短发夹RNA的慢病毒载体转染小鼠视网膜的干扰效率研究

Efficiency of targeted interference on early growth response factor-1 by lentivirus vector containing shRNA transfected into mouse retina

摘要:

目的 构建携带绿色荧光蛋白(GFP)和靶向干扰早期生长反应基因1(Egr-1)短发夹RNA(shRNA)共表达的慢病毒载体,转染小鼠视网膜组织,观察其对Egr-1基因的干扰效率.方法 针对已经筛选确定的Egr-1基因shRNA有效靶序列,构建携带GFP和靶向干扰Egr-1 shRNA的慢病毒载体LV-shRNA(Egr-1).15日龄C57BL/6小鼠完全随机法分为实验组和阴性对照组,每组10只.LVshRNA(Egr-1)慢病毒载体经玻璃体腔注射转染至实验组小鼠右眼内,不针对任何特异基因的LV-NC 慢病毒载体通过同样的转染途径转染至阴性对照组小鼠右眼内,实验组及阴性对照组小鼠左眼不做任何处理设为空白对照组.2周后荧光显微镜下观察转染情况,实时定量PCR(RQ-PCR)、免疫印迹(Western blot)、免疫荧光检测Egr-1的表达,观察Egr-1基因的干扰效率.结果 慢病毒载体经玻璃体腔注射途径转染小鼠视网膜后,GFP广泛分布于视网膜全层,包括视网膜色素上皮层.与空白对照组和阴性对照组注射眼比较,RQ-PCR检测显示实验组注射眼Egr-1 mRNA表达明显下调(0.290+0.074比1.006±0.033、1.010±0.086,均P<0.001),抑制率为71.29%;免疫印迹显示实验组注射眼内Egr-1蛋白表达明显下调(0.224±0.035比0.674±0.050、0.688±0.049,P<0.001),抑制率为67.44%.免疫荧光检测发现实验组注射眼在视网膜除内核层有少许Egr-1阳性细胞分布外,没有荧光表达.结论 成功构建携带绿色荧光蛋白和靶向干扰Egr-1基因shRNA的慢病毒载体,经玻璃体腔注射转染小鼠眼内其转染效率高,分布范围广,且对小鼠视网膜Egr-1基因抑制效率高.

更多
abstracts:

Objective To construct the lentivirus vector containing green fluorescent protein (GFP)and early growth response factor-1 short hairpin RNA(Egr-1 shRNA),and to determine the efficiency of interference on Egr-1 gene in lentivirus vector-transfected mouse retinal tissue.Methods Based on the target sequences of Egr-1 shRNA screened and identified previously,the LV-shRNA(Egr-1)containing GFP and Egr-1 shRNA Was constructed.Twenty 15-day-old C57BL/6 mice were randomly divided into experimental group and negative control group(n=10 each).LV-shRNA(Egr-1)lentivirus vector Was transfected into the right eyes in the experimental group with intravitreal injection.LV-NC lentivirus vector that did not target at any specific gene Was also transfected into the right eyes in the negative control group,while the left eyes were untreated in both two groups as the blank control group.After 2 weeks,the transfection of lentiviral vector was examined under fluorescence microscope.Moreover,real-time quantitative polymerase chain reaction (RQ-PCR), Western blot and immunofluorescence were used to detect the expression of Egr-1 gene for evaluation of the interference efficiency. Results After lentiviral vector was intravitreally injected and transfected into the mouse retinal tissue, GFP was found to be widely distributed in the whole layer of the retina including the retinal pigment epithelium. RQPCR showed significantly down-regulated expression of Egr-1 mRNA in the injected eyes of experimental group compared with blank or negative controls (0.290±0.074 vs 1.006±0.033, 1.010± 0.086, all P<0.001) , with the inhibition rate being 71.29%. Western blot demonstrated that the expression of Egr-1 protein was remarkably down-regulated in the injected eyes of experimental group (0.224±0.035 vs 0.674±0.050, 0.688±0.049, all P<0.00l) , with the inhibition rate being 67.44%. Furthermore, immunofluorescence revealed no expression of fluorescence protein, except sparse distribution of Egr-1 positive cells in the inner nuclear layer of injected eyes of the experimental group. Conclusions The lentivirus vector containing GFP and Egr-1 shRNA is successfully constructed, and it may have high transfection efficiency and a wide distribution in the intravitreally injected mice eyes. Furthermore, the lentivirus vector has a high inhibition efficiency of Egr-1 gene in the mouse retina.

More
作者: 蒋晶晶 [1] 刘双珍 [1] 文丹 [1] 王沙 [1]
分类号: R378.2
栏目名称: 论著
DOI: 10.3760/cma.j.issn.1674-1927.2011.02.004
发布时间: 2011-09-08
基金项目:
湖南省自然科学基金 湖南省科技厅重点项目
  • 浏览:302
  • 下载:68

加载中!

相似文献

  • 中文期刊
  • 外文期刊
  • 学位论文
  • 会议论文

加载中!

加载中!

加载中!

加载中!

扩展文献

特别提示:本网站仅提供医学学术资源服务,不销售任何药品和器械,有关药品和器械的销售信息,请查阅其他网站。

充值 订阅 收藏 移动端 使用
帮助