Feuer G, Fan H
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
J Virol. 1990 Dec;64(12):6130-40. doi: 10.1128/JVI.64.12.6130-6140.1990.
The effects of inserting cellular regulatory sequences from the murine transthyretin (TTR) gene into the Moloney murine leukemia virus (M-MuLV) long terminal repeat (LTR) were investigated. Transthyretin is expressed predominantly in the liver and choroid plexus in adult mice, and TTR upstream regulatory elements were previously shown to potentiate transcription in liver-derived cells. The effects of inserting the TTR distal enhancer and/or promoter-proximal sequences into an M-MuLV LTR lacking its enhancers were measured in three ways. (i) Chimeric LTRs were fused to the bacterial chloramphenicol acetyltransferase gene (cat) and tested for transient gene expression by transfection into liver-derived cells or NIH 3T3 fibroblasts. (ii) Infectious M-MuLV containing an altered LTR [delta Mo + TTR(PD) MuLV) was generated, and infectivity in culture on hepatocyte lines and NIH 3T3 cells was tested. (iii) Infection of delta Mo + TTR(PD) MuLV in vivo was tested by inoculating NFS/N mice and performing in situ hybridization of whole animal sections. Chimeric LTR-cat constructs showed higher levels of cat gene expression in liver-derived cell lines than in NIH 3T3 cells, indicating increased LTR activity in these cells. However, in vitro infection did not show significantly higher infectivity in hepatocytes for delta Mo + TTR(PD) M-MuLV than did wild-type M-MuLV. In vivo, delta Mo + TTR(PD) MuLV showed expression in the same tissues as with wild-type M-MuLV-inoculated mice, i.e., lymphoid organs and the intestines and, additionally, two novel sites not seen in wild-type M-MuLV-inoculated animals. Of 10 mice, 8 showed viral expression in the brain and 3 showed expression in the liver. Thus, insertion of TTR elements into the M-MuLV LTR altered LTR activity both in vitro and in vivo.
研究了将小鼠甲状腺素运载蛋白（TTR）基因的细胞调节序列插入莫洛尼氏鼠白血病病毒（M-MuLV）长末端重复序列（LTR）的效果。甲状腺素运载蛋白在成年小鼠的肝脏和脉络丛中主要表达，并且TTR上游调节元件先前已显示可增强肝源性细胞中的转录。通过三种方式测量了将TTR远端增强子和/或启动子近端序列插入缺乏其增强子的M-MuLV LTR中的效果。（i）将嵌合LTR与细菌氯霉素乙酰转移酶基因（cat）融合，并通过转染到肝源性细胞或NIH 3T3成纤维细胞中测试瞬时基因表达。（ii）产生含有改变的LTR [δMo + TTR（PD）MuLV]的感染性M-MuLV，并测试其在肝细胞系和NIH 3T3细胞上的培养感染性。（iii）通过接种NFS/N小鼠并对整个动物切片进行原位杂交来测试δMo + TTR（PD）MuLV在体内的感染情况。嵌合LTR-cat构建体在肝源性细胞系中显示出比NIH 3T3细胞更高水平的cat基因表达，表明这些细胞中LTR活性增加。然而，在体外感染中，δMo + TTR（PD）M-MuLV在肝细胞中的感染性并未比野生型M-MuLV显著更高。在体内，δMo + TTR（PD）MuLV与野生型M-MuLV接种小鼠一样，在相同组织中表达，即淋巴器官和肠道，此外，在野生型M-MuLV接种动物中未见的两个新位点也有表达。在10只小鼠中，8只在脑中显示病毒表达，3只在肝脏中显示表达。因此，将TTR元件插入M-MuLV LTR在体外和体内均改变了LTR活性。
