Strict control of transgene expression in a mouse model for sensitive biological applications based on RMCE compatible ES cells.
Strict control of transgene expression in a mouse model for sensitive biological applications based on RMCE compatible ES cells.
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DOI：
10.1093/nar/gkq868
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发表时间：
2011-01
影响因子：
14.9
通讯作者：
Wirth D
中科院分区：
文献类型：
--
作者：
Sandhu U;Cebula M;Behme S;Riemer P;Wodarczyk C;Metzger D;Reimann J;Schirmbeck R;Hauser H;Wirth D
具有严格控制的转基因表达的重组小鼠品系被证明是阐明基因功能的不可或缺的工具。已经采用不同的策略来实现转基因的受控诱导。然而，许多模型在非诱导状态下伴随着相当高水平的基础表达。因此，排除了要求严格控制转基因表达的应用，例如毒性基因的表达和对neo抗原的免疫应答的研究。我们开发了一种新的Cre/loxP为基础的策略，以实现严格控制转基因表达。该策略与RMCE（重组酶介导的盒交换）相结合，该RMCE促进基因靶向ES细胞中的标记位点。使用荧光素酶作为报告基因证实了调控的紧密性。在将这些小鼠培育成具有CreERT 2的普遍（ROSA 26）或肝脏特异性（白蛋白）表达的效应动物，随后用他莫昔芬喂养时诱导转基因。利用RMCE技术，用卵清蛋白抗原替代荧光素酶。由这些ES细胞产生的小鼠与表达肝脏特异性CreERT 2的小鼠交配。检查转基因小鼠是否建立免疫应答。它们完全有能力在肝细胞特异性OVA抗原表达后建立免疫应答，如他莫昔芬治疗后大量肝损伤所示，并且没有显示出OVA耐受性。总之，这证明了这种策略支持严格控制转基因，甚至与高度敏感的生物读数兼容。
Recombinant mouse strains that harbor tightly controlled transgene expression proved to be indispensible tools to elucidate gene function. Different strategies have been employed to achieve controlled induction of the transgene. However, many models are accompanied by a considerable level of basal expression in the non-induced state. Thereby, applications that request tight control of transgene expression, such as the expression of toxic genes and the investigation of immune response to neo antigens are excluded. We developed a new Cre/loxP-based strategy to achieve strict control of transgene expression. This strategy was combined with RMCE (recombinase mediated cassette exchange) that facilitates the targeting of genes into a tagged site in ES cells. The tightness of regulation was confirmed using luciferase as a reporter. The transgene was induced upon breeding these mice to effector animals harboring either the ubiquitous (ROSA26) or liver-specific (Albumin) expression of CreERT2, and subsequent feeding with Tamoxifen. Making use of RMCE, luciferase was replaced by Ovalbumin antigen. Mice generated from these ES cells were mated with mice expressing liver-specific CreERT2. The transgenic mice were examined for the establishment of an immune response. They were fully competent to establish an immune response upon hepatocyte specific OVA antigen expression as indicated by a massive liver damage upon Tamoxifen treatment and did not show OVA tolerance. Together, this proves that this strategy supports strict control of transgenes that is even compatible with highly sensitive biological readouts.
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影响因子：
4.6
作者：
Hameyer, Dorothe;Loonstra, Ate;Bockamp, Ernesto
通讯作者：
Bockamp, Ernesto
影响因子：
14.9
作者：
Indra, AK;Warot, X;Metzger, D
通讯作者：
Metzger, D
DOI：
10.1006/bbrc.1997.7124
发表时间：
1997-08-28
影响因子：
3.1
作者：
Feil, R;Wagner, J;Chambon, P
通讯作者：
Chambon, P
影响因子：
4.8
作者：
Manickan, E;Satoi, JJ;Liang, TJ
通讯作者：
Liang, TJ
影响因子：
3.5
作者：
Nehlsen K;Schucht R;da Gama-Norton L;Krömer W;Baer A;Cayli A;Hauser H;Wirth D
通讯作者：
Wirth D