Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon.
Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon.
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DOI：
10.1186/1472-6750-6-30
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发表时间：
2006-06-26
影响因子：
3.5
通讯作者：
Largaespada DA
中科院分区：
文献类型：
--
作者：
Geurts AM;Wilber A;Carlson CM;Lobitz PD;Clark KJ;Hackett PB;McIvor RS;Largaespada DA
转座因子插入突变技术已成为研究模式生物的遗传学家们的热门技术。从大肠杆菌到果蝇，以及最近的老鼠。其中一个这样的元件是睡美人（SB）转座子，它在几项研究中已被证明是小鼠生殖系中有效的插入诱变剂。SB转座子载体研究已经采用不同的功能元件和报告分子来破坏和报告内源小鼠基因的表达。我们试图产生一种转座子系统，该系统能够报告小鼠基因的表达模式，同时允许感兴趣的基因以组织或时间特异性模式的条件表达。在这里，我们报告了系统的开发和测试的转座子为基础的基因陷阱系统，结合强力霉素抑制Tet-Off（tTA）系统，能够激活基因的表达控制下的泰特响应元件（TRE）启动子。我们证明，基因陷阱系统是完全功能性的体外引入的“基因陷阱tTA”载体到人类细胞中的转座和鉴定克隆，激活表达的一个TRE-fluorescence转基因的强力霉素依赖性的方式。在转基因小鼠中，我们动员基因陷阱tTA载体，发现可以影响生殖系动员率的参数，并确定候选基因插入，以证明载体系统的体内功能。我们进一步证明，基因陷阱可以作为内源性基因表达的报告，它可以与生物发光成像，以确定基因的组织特异性表达模式。类似于果蝇中使用的GAL 4/UAS系统，我们已经取得了进展，开发了一种用于突变和揭示小鼠基因表达的工具，通过在二级TRE-regulated报告分子存在下产生tTA反式激活因子。一个载体，如基因陷阱tTA可以提供一种手段，既注释小鼠基因，并创建一个资源的小鼠表达的时间和组织特异性模式的可调控的转录因子的条件基因表达研究。这些小鼠将是一个宝贵的资源，小鼠遗传学界的目的解剖哺乳动物基因功能。
Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB) transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern. Here we report the systematic development and testing of a transposon-based gene-trap system incorporating the doxycycline-repressible Tet-Off (tTA) system that is capable of activating the expression of genes under control of a Tet response element (TRE) promoter. We demonstrate that the gene trap system is fully functional in vitro by introducing the "gene-trap tTA" vector into human cells by transposition and identifying clones that activate expression of a TRE-luciferase transgene in a doxycycline-dependent manner. In transgenic mice, we mobilize gene-trap tTA vectors, discover parameters that can affect germline mobilization rates, and identify candidate gene insertions to demonstrate the in vivo functionality of the vector system. We further demonstrate that the gene-trap can act as a reporter of endogenous gene expression and it can be coupled with bioluminescent imaging to identify genes with tissue-specific expression patterns. Akin to the GAL4/UAS system used in the fly, we have made progress developing a tool for mutating and revealing the expression of mouse genes by generating the tTA transactivator in the presence of a secondary TRE-regulated reporter molecule. A vector like the gene-trap tTA could provide a means for both annotating mouse genes and creating a resource of mice that express a regulable transcription factor in temporally- and tissue-specific patterns for conditional gene expression studies. These mice would be a valuable resource to the mouse genetics community for purpose of dissecting mammalian gene function.
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影响因子：
48
作者：
Keng, VW;Yae, K;Takeda, J
通讯作者：
Takeda, J
影响因子：
4.4
作者：
Adams, DJ;Quail, MA;Bradley, A
通讯作者：
Bradley, A
影响因子：
14.9
作者：
Krestel, H. E.;Mayford, M. R.;Sprengel, R.
通讯作者：
Sprengel, R.
影响因子：
64.5
作者：
Ivics, Z;Hackett, PB;Izsvak, Z
通讯作者：
Izsvak, Z
DOI：
10.1186/1477-7827-2-73
发表时间：
2004-10-17
期刊：
Reproductive biology and endocrinology : RB&E
影响因子：
--
作者：
Hynninen, Piritta;Hamalainen, Jonna M;Parkkila, Seppo
通讯作者：
Parkkila, Seppo