Sander A, Güth A, Brenner H R, Witzemann V
Abteilung Zellphysiologie, Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany.
Cell Tissue Res. 2000 Sep;301(3):397-403. doi: 10.1007/s004410000247.
Pressure injection of DNA directly into individual fibers of surgically exposed soleus muscle leads to efficient and reliable expression of the transgene. Conditionally regulated gene expression in a single muscle fiber was analyzed in vivo by co-injecting a tetracycline-regulated lacZ reporter construct and a transactivator (rtTA) expression vector. The tetracycline-responsive element revealed significant basal transcriptional activity that was further increased by rtTA even in the absence of the effector doxycycline (dox). The high basal activity of the simple two-component system precludes tight gene regulation in muscle. Concomitant expression of the silencer tTS(Kid), however, reduced the basal activity to low or undetectable levels. This allowed the specific activation of the tetracycline-responsive element by the application of dox. Direct gene transfer can thus be employed to express transgenic proteins in distinct muscle fibers at spatially defined regions and to regulate gene expression conditionally.
将DNA直接压力注射到手术暴露的比目鱼肌的单个肌纤维中可导致转基因高效且可靠地表达。通过共注射四环素调控的lacZ报告基因构建体和反式激活因子（rtTA）表达载体，在体内分析单个肌纤维中的条件性调控基因表达。四环素反应元件显示出显著的基础转录活性，即使在没有效应物强力霉素（dox）的情况下，rtTA也能进一步增强这种活性。简单的双组分系统的高基础活性妨碍了肌肉中基因的严格调控。然而，沉默子tTS（Kid）的共表达将基础活性降低到低水平或无法检测的水平。这使得通过应用dox可特异性激活四环素反应元件。因此，直接基因转移可用于在空间限定区域的不同肌纤维中表达转基因蛋白，并可条件性地调控基因表达。
