First inducible transgene expression in porcine large animal models
First inducible transgene expression in porcine large animal models
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DOI：
10.1096/fj.11-185041
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发表时间：
2012-03-01
期刊：
影响因子：
4.8
通讯作者：
Wolf, Eckhard
中科院分区：
文献类型：
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作者：
Klymiuk, Nikolai;Boecker, Wolfgang;Wolf, Eckhard
本研究的目的是建立诱导转基因表达的猪，一个模式生物与实验生理学和转化医学的巨大希望，使用二元tet-on系统。该表达系统由强力霉素（dox）通过tet控制的反式激活因子（TA）激活。TA与反式激活因子反应元件（TRE）的结合导致下游基因的转录。首先，我们克隆了在CMV增强子/鸡β-肌动蛋白启动子（CAG）控制下表达TA的转基因创始猪。然后，将来自CAG-TA转基因建立者的细胞用针对猪细胞毒性T淋巴细胞相关抗原4-Fc结构域免疫球蛋白G1（CTLA-4 Ig）或可溶性NF-κ B配体受体激活剂（RANKL）的TRE-controlled表达载体进行核转染，并克隆双转基因后代。在核转染细胞和转基因猪中，Dox给药导致CTLA-4 Ig或RANKL表达呈剂量依赖性增加，而在没有Dox的情况下，两种蛋白质的水平均低于检测限。通过克隆或育种产生的双转基因后代中再现了诱导型转基因表达。我们的策略揭示了猪中诱导型转基因表达的前两个例子。本研究中产生的CAG-TA转基因猪为未来具有诱导型转基因表达的猪模型奠定了有趣的基础。Klymiuk，N.，Bocker，W.，Schonitzer，V.，Bahr，A.，Radic，T.，Frohlich，T.，Wunsch，A.，Kessler，B.，Kurome，M.，Schilling，E.，Herbach，N.，万科河，Nagashima，H.，Mutschler，W.，Arnold，G. J.，Schwinzer河，Schieker，M.，沃尔夫，E.在猪大型动物模型中首次诱导转基因表达。FASEB J.26，1086-1099（2012）。www.fasebj.org
The purpose of this study was to establish inducible transgene expression in pigs, a model organism with great promise for experimental physiology and translational medicine, using the binary tet-on system. This expression system is activated by doxycycline (dox) via the tet-controlled transactivator (TA). Binding of TA to the transactivator response element (TRE) results in transcription of downstream genes. First, we cloned transgenic founder pigs expressing TA under the control of the CMV enhancer/chicken beta-actin promoter (CAG). Then, cells from CAG-TA transgenic founders were nucleofected with TRE-controlled expression vectors for either porcine cytotoxic T-lymphocyte associated antigen 4-Fc domain of immunoglobulin G1 (CTLA-4Ig) or soluble receptor activator of NF-kappa B ligand (RANKL), and double-transgenic offspring were cloned. Dox administration resulted in a dose-dependent increase in expression of CTLA-4Ig or RANKL, in nucleofected cells and in transgenic pigs, while in the absence of dox, the levels of both proteins were below the detection limit. Inducible transgene expression was reproduced in double-transgenic offspring generated by cloning or breeding. Our strategy revealed the first two examples of inducible transgene expression in pigs. The CAG-TA transgenic pigs generated in this study constitute an interesting basis for future pig models with inducible transgene expression.- Klymiuk, N., Bocker, W., Schonitzer, V., Bahr, A., Radic, T., Frohlich, T., Wunsch, A., Kessler, B., Kurome, M., Schilling, E., Herbach, N., Wanke, R., Nagashima, H., Mutschler, W., Arnold, G. J., Schwinzer, R., Schieker, M., Wolf, E. First inducible transgene expression in porcine large animal models. FASEB J. 26, 1086-1099 (2012). www.fasebj.org