TISSUE LOCALIZATION OF SIV EXPRESSING GREEN FLUORESCENT PROTEIN

SIV的一般致病机制研究及早期鉴定 特别是病毒感染的目标细胞将大大 通过容易地识别感染细胞的技术来促进 为此，微生物部与 病理分部已经构建了一种重组病毒，能够 增强型绿色荧光蛋白在体内的表达 之所以被选中，是因为该基因很小(717个碱基)，允许准备好 插入到SIVmac239的适当缺失突变体中 插入到SIVmac239的一个变异体的nef基因座，该变异体包含一个 Nef基因的大量缺失产生了一种复制的病毒 在CEMx174细胞中快速表达并表达EGFP，导致 在不添加任何发色原的情况下，细胞的荧光强度超过90% 当p27Gag表达达到 其高峰EGFP在该细胞系中的表达保持稳定 通过多代重组病毒检测 体内免疫表型病毒阳性细胞，我们已经修改了我们的 现有的密码保护技术以增强GFP和 与抗肿瘤单抗结合的二次染色质 体内细胞表位我们在CD4+中检测到丰富的病毒 外周淋巴结内淋巴细胞与CFA肉芽肿 感染动物的时间范围与未观察到的类似 表达绿色荧光蛋白的病毒出版物亚历山大，L，Veazey RS，沙国S， DeMaria M，Rosenzweig M，Lackner AA，Desrosiers RC，VG萨塞维尔 表达绿色荧光的重组猿猴免疫缺陷病毒 蛋白质识别恒河猴艾滋病病毒感染细胞 逆转录病毒15：11-21,1999

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