Weber Wilfried, Marty René R, Link Nils, Ehrbar Martin, Keller Bettina, Weber Cornelia C, Zisch Andreas H, Heinzen Christoph, Djonov Valentin, Fussenegger Martin
Institute of Biotechnology, Swiss Federal Institute of Technology Zurich, ETH Hoenggerberg, CH-8093 Zurich, Switzerland.
Nucleic Acids Res. 2003 Jun 15;31(12):e69. doi: 10.1093/nar/gng069.
Advanced heterologous transcription control systems for adjusting desired transgene expression are essential for gene function assignments, drug discovery, manufacturing of difficult to produce protein pharmaceuticals and precise dosing of gene-based therapeutic interventions. Conversion of the Streptomyces albus heat shock response regulator (RheA) into an artificial eukaryotic transcription factor resulted in a vertebrate thermosensor (CTA; cold-inducible transactivator), which is able to adjust transcription initiation from chimeric target promoters (P(CTA)) in a low-temperature- inducible manner. Evaluation of the temperature-dependent CTA-P(CTA) interaction using a tailored ELISA-like cell-free assay correlated increased affinity of CTA for P(CTA) with temperature downshift. The temperature-inducible gene regulation (TIGR) system enabled tight repression in the chicken bursal B-cell line DT40 at 41 degrees C as well as precise titration of model product proteins up to maximum expression at or below 37 degrees C. Implantation of microencapsulated DT40 cells engineered for TIGR-controlled expression of the human vascular endothelial growth factor A (hVEGF121) provided low-temperature-induced VEGF-mediated vascularization in chicken embryos.
用于调节所需转基因表达的先进异源转录控制系统对于基因功能分配、药物发现、难以生产的蛋白质药物制造以及基于基因的治疗干预的精确给药至关重要。将白色链霉菌热休克反应调节因子（RheA）转化为人工真核转录因子，产生了一种脊椎动物热传感器（CTA；冷诱导反式激活因子），它能够以低温诱导的方式调节嵌合靶启动子（P(CTA)）的转录起始。使用定制的类似ELISA的无细胞测定法评估温度依赖性CTA-P(CTA)相互作用，发现CTA对P(CTA)的亲和力增加与温度下降相关。温度诱导基因调控（TIGR）系统能够在41℃时对鸡法氏囊B细胞系DT40进行严格抑制，并能精确滴定模型产物蛋白，直至在37℃或以下达到最大表达。植入经工程改造用于TIGR控制人血管内皮生长因子A（hVEGF121）表达的微囊化DT40细胞，可在鸡胚中提供低温诱导的VEGF介导的血管生成。
