An inducible and reversible mouse genetic rescue system.
An inducible and reversible mouse genetic rescue system.
复制标题
DOI：
10.1371/journal.pgen.1000069
复制
发表时间：
2008-05-09
期刊：
影响因子：
4.5
通讯作者：
Gaitanaris, George A.
中科院分区：
文献类型：
--
作者：
Zeng, Hongkui;Horie, Kyoji;Madisen, Linda;Pavlova, Maria N.;Gragerova, Galina;Rohde, Alex D.;Schimpf, Brian A.;Liang, Yuqiong;Ojala, Ethan;Kramer, Farah;Roth, Patricia;Slobodskaya, Olga;Dolka, Io;Southon, Eileen A.;Tessarollo, Lino;Bornfeldt, Karin E.;Gragerov, Alexander;Pavlakis, George N.;Gaitanaris, George A.
基因表达的诱导性和可逆性调控是揭示基因功能的有效方法。我们已经建立了一种在小鼠中有效产生可逆和诱导基因敲除和拯救的通用方法。在这个我们命名为 iKO 的系统中，通过用强力霉素治疗小鼠，可以随意打开和关闭目标基因。该方法结合了两种转基因小鼠品系：a) 带有四环素依赖性反式激活因子的 KO 品系，取代了内源靶基因；b) 带有插入严格调控 (TIGRE) 基因组位点的四环素诱导型靶基因 cDNA 的品系，该品系提供了低基础表达和高诱导性。这样的位点在基因组中很少出现，我们开发了一种通过重组酶介导的插入轻松将基因引入小鼠胚胎干（ES）细胞的 TIGRE 位点的方法。 KO 和 TIGRE 品系均专为高通量、大规模且经济高效的 iKO 小鼠生产而设计。作为概念证明，我们在载脂蛋白 E (ApoE) 基因中创建了 iKO 小鼠，可进行灵敏且定量的表型分析。结果表明 ApoE 转录、血浆胆固醇水平以及动脉粥样硬化进展和消退的可逆转换。 iKO系统显示出严格的监管，是一种多功能遗传系统，可以轻松整合其他技术并适应广泛的应用。我们描述了一种在小鼠中产生可诱导和可逆基因失活的技术。它结合了两种转基因小鼠品系：一种是用四环素反式激活子取代内源靶基因的敲除品系，另一种是四环素诱导的靶基因 cDNA 已插入特定基因组位点的品系。该系统的一个关键组成部分是我们已识别和设计的独特染色体位点，它提供了一个平台，可以轻松插入任何感兴趣的基因以严格控制表达。由于其简单的二元性质，允许对两个组件中的每一个进行独立修改，并且可以在高通量模式下使用，我们相信我们的系统将可用于多种应用，例如引入靶基因的突变体或人源化形式以及功能操作工具。我们已将此技术应用于载脂蛋白 E (ApoE) 基因，并证明：a) ApoE 的表达严格依赖于小鼠饮食中强力霉素（一种四环素类抗生素）的存在，b) 在缺乏强力霉素（ApoE 抑制）的情况下，动脉粥样硬化斑块形成，证实了 ApoE 在此过程中的重要性，以及 c) 在小鼠体内重新诱导 ApoE 时，多西环素，动脉粥样硬化消退。
Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications. We describe a technology for the creation of inducible and reversible gene inactivation in mice. It combines two genetically modified mouse lines: a knock-out line with a tetracycline transactivator replacing the endogenous target gene, and a line in which a tetracycline-inducible cDNA of the target gene has been inserted into a specific genomic locus. A critical component of this system is the unique chromosomal loci we have identified and engineered that offer a platform for easy insertion of any gene of interest for tightly controlled expression. Because of its simple binary nature, allowing independent modification of each of the two components and possibility of use in a high-throughput mode, we believe that our system will be useful for multiple applications, such as introducing mutant or humanized form of the target gene as well as functional manipulating tools. We have applied this technology to the Apolipoprotein E (ApoE) gene and have demonstrated that: a) the expression of ApoE is strictly dependent on the presence of doxycycline, a tetracycline group antibiotic, in the mouse diet, b) in the absence of doxycycline (ApoE repressed) atherosclerotic plaques are formed, confirming the importance of ApoE in the process, and c) upon re-induction of ApoE in the animals with doxicyclin, atherosclerosis regressed.
登录
查看更多内容
影响因子：
3.6
作者：
Chen, JS;Kelz, MB;Nestler, EJ
通讯作者：
Nestler, EJ
影响因子：
46.9
作者：
Fussenegger, M;Morris, RP;Bailey, JE
通讯作者：
Bailey, JE
影响因子：
3.5
作者：
Anastassiadis, K;Kim, J;Stewart, AF
通讯作者：
Stewart, AF
DOI：
10.1073/pnas.93.8.3346
发表时间：
1996-04-16
影响因子：
11.1
作者：
No, D;Yao, TP;Evans, RM
通讯作者：
Evans, RM
影响因子：
56.9
作者：
Bond, CT;Sprengel, R;Adelman, JP
通讯作者：
Adelman, JP