In vivo Regulation of the Cell Fates Utilizing a Novel Inducible System of Protein Expression

按照计划，我构建了可诱导的逆转录病毒系统，该系统可以对目标蛋白的表达进行快速和剂量相关的调节。然而，事实证明，降解域(DD)/Shield-1系统本身确实存在内在缺陷，这与显著的表达泄漏是相容的。因此，我决定改变齿轮，结合使用DD/Shield-1和四环素诱导系统，后者最初是由Bujard博士开发的。我使用了所谓的“all -in- one LV”，这是我的同事制作的慢病毒系统，它可以在一个卡带中包含系统工作所需的所有元素。在组合测试中，我最终发现由Tet-ON和DD/Shield-1组成的系统性能最好，因为它可以实现最低的泄漏和双重增产时的快速表达。虽然由于计划的改变，总体进展仍落后于计划，但我相信，一旦在这种新开发的双调控慢病毒载体中引入可诱导的靶基因，该系统现在已经准备好进行体内测试。

As planned, I constructed the inducible retroviral system that could allow rapid and dose-related regulation of expression of the target protein. It, however, turned out that the degradation-domain(DD)/Shield-1 system did have an intrinsic defect in itself, which was compatible with significant expression leak. I thus decided to change gears to the combined use of DD/Shield-1 and tetracycline-inducible system, the latter of which was originally developed by Dr. Bujard. I utilized so-called "All-in-One LV", the lentiviral system that was generated by my colleague to allow containing all the required elements for the system to work within a single cassette. Among the combination tested, I finally figured out that the system comprised of Tet-ON and DD/Shield-1 was the best performer so that it allowed the lowest leak and prompt expression upon dual stimulation. Although the overall progress remain behind the schedule due to the change of plans, I believe that the system is now ready to be tested in vivo, once the inducible target gene is introduced within this newly developed dual-regulation lentiviral vector.