Rapid retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette
Rapid retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette
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DOI：
10.1073/pnas.93.11.5185
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发表时间：
1996-05-28
影响因子：
11.1
通讯作者：
Blau, HM
中科院分区：
文献类型：
--
作者：
Hofmann, A;Nolan, GP;Blau, HM
我们描述了一个单一的自动调节盒，允许可逆诱导的转基因表达响应四环素（泰特）。该盒含有先前由其他人描述的在两个单独质粒上的所有必需组分，所述质粒在数月内顺序引入[Gossen，M. & Bujard，H.等人（1992）Proc. Acad. Sci. USA 89，5547-5551]。使用逆转录病毒引入盒，允许转移到难以转染的细胞类型中。因此，数千个细胞的群体，而不是几个克隆，可以在几周内分离和表征。为了避免强逆转录病毒长末端重复序列增强子和启动子元件对tet调节的巨细胞病毒最小启动子功能的潜在干扰，载体是自失活的，在感染靶细胞后消除长末端重复序列的转录。串联tet操纵子序列和巨细胞病毒最小启动子驱动双顺反子mRNA的表达，导致目的基因（lacZ）和内部核糖体进入位点控制的反式激活因子（泰特阻遏物-VP 16融合蛋白）的转录。在没有泰特的情况下，反式激活因子通过自动调节环进行性增加，而在泰特存在的情况下，基因表达被阻止。北方印迹、生物化学和单细胞分析均显示该构建体产生低基础水平的基因表达和一到两个数量级的诱导。因此，目前的盒的逆转录病毒构建体（SIN-RetroTet载体）允许诱导基因的快速交付，并应具有广泛的应用培养细胞，转基因动物，和基因治疗。
We describe a single autoregulatory cassette that allows reversible induction of transgene expression in response to tetracycline (tet). This cassette contains all of the necessary components previously described by others on two separate plasmids that are introduced sequentially over a period of months [Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. The cassette is introduced using a retrovirus, allowing transfer into cell types that are difficult to transfect. Thus, populations of thousands of cells, rather than a few clones, can be isolated and characterized within weeks. To avoid potential interference of the strong retroviral long terminal repeat enhancer and promoter elements with the function of the tet-regulated cytomegalovirus minimal promoter, the vector is self-inactivating, eliminating transcription from the long terminal repeat after infection of target cells. Tandem tet operator sequences and the cytomegalovirus minimal promoter drive expression of a bicistronic mRNA, leading to transcription of the gene of interest (lacZ) and the internal ribosome entry site controlled transactivator (Tet repressor-VP16 fusion protein). In the absence of tet, there is a progressive increase in transactivator by means of an autoregulatory loop, whereas in the presence of tet, gene expression is prevented. Northern blot, biochemical, and single cell analyses have all shown that the construct yields low basal levels of gene expression and induction of one to two orders of magnitude. Thus, the current cassette of the retroviral construct (SIN-RetroTet vector) allows rapid delivery of inducible genes and should have broad applications to cultured cells, transgenic animals, and gene therapy.