TISSUE LOCALIZATION OF SIV EXPRESSING GREEN FLUORESCENT PROTEIN

表达绿色荧光蛋白的 SIV 的组织定位

• 批准号: 6453786
• 负责人: VITO G SASSEVILLE
• 金额: $ 11.11万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6453786
• 关键词: AIDS; Mammalia; animal tissue; communicable diseases; hematology; immunology; inflammation; lymphatic system; skin; virus

Studies of SIV pathogenesis in general and identification of early target cells of viral infection in particular would be greatly facilitated by techniques to readily identify infected cells Toward this end, the Microbiology Division in collaboration with the Pathology Division has constructed a recombinant virus that is capable of expressing enhanced green fluorescent protein (EGFP) in vivo EGFP was selected because the gene is small (717 bp) allowing for ready insertion into appropriate deletion mutants of SIVmac239 The insertion of EGFP into the nef locus of a variant of SIVmac239 that contained a large deletion in the nef gene produced a virus that replicated rapidly in CEMx174 cells and expressed EGFP resulting in strong fluorescence of the cells without addition of any chromogen Over 90% of the cultured cells expressed EGFP when p27 gag expression reached its peak The expression of EGFP remained stable in this cell line through multiple passages of th e recombinant virus To detect the immunophenotype virus-positive cells in vivo, we have modified our existing cryppreservation techniques to enhance colabeling of GFP and a secondary chromagen bound to monoclonal antibodies directed against cellular epitopes In vivo we have detected abundant virus in CD4+ lymphocytes within peripheral lymph nodes and CFA granulomas in infected animals in a time frame similar to that observed with non EGFP-expressing virus Publication Alexander, L, Veazey RS, Czajak S, DeMaria M, Rosenzweig M, Lackner AA, Desrosiers RC, Sasseville, VG Recombinant simiam immunodeficiency virus expressing green fluorescent protein identifies infected cells in rhesus monkeys AIDS Res Hum Retroviruses 15:11-21, 1999

SIV发病机理的一般研究和早期鉴定 尤其是病毒感染的靶细胞将是极大的 通过技术促进，容易识别受感染的细胞朝向 这是微生物学部与 病理分裂已经建立了一种能够的重组病毒 在体内表达增强的绿色荧光蛋白（EGFP） 之所以选择，是因为该基因很小（717 bp）允许准备就绪 插入SIVMAC239的适当缺失突变体中插入 EGFP的sivmac239变体的NEF基因座，其中包含一个 NEF基因中的大删除产生了复制的病毒 在CEMX174细胞中迅速表达EGFP，导致强大 细胞的荧光，而无需添加任何超过90％的发色原 培养的细胞表达p27 GAG表达时表达EGFP 它的峰值EGFP的表达在此细胞系中保持稳定 通过重组病毒的多个通道检测 免疫表型病毒阳性细胞在体内，我们修改了我们的 现有的哭泣技术来增强GFP和 与针对单克隆抗体的二级染色体 细胞表位在体内我们发现了CD4+的大量病毒 外周淋巴结和CFA肉芽肿内的淋巴细胞 在一个时间框架中，感染动物类似于未观察到的动物 EGFP表达病毒出版物Alexander，L，Veazey RS，Czajak S， Demaria M，Rosenzweig M，Lackner AA，Desrosiers RC，Sasseville，VG 表达绿色荧光的重组Simiam免疫缺陷病毒 蛋白质鉴定恒河猴中受感染的细胞aids res hum 逆转录病毒15：11-21，1999