Harding T C, Geddes B J, Noel J D, Murphy D, Uney J B
Department of Medicine, University of Bristol, England, U.K.
J Neurochem. 1997 Dec;69(6):2620-3. doi: 10.1046/j.1471-4159.1997.69062620.x.
A transfer system that enabled the efficient introduction of transgenes into neurones and the quantitative control of the expressed transgene would greatly facilitate studies into neuronal gene function. To develop such a system we incorporated the tetracycline (Tet)-responsive On/Off regulatory elements into type-5 adenoviral (Ad) vectors. Regulation of transgene expression following transfection was measured by placing the enhanced green fluorescent protein (EGFP) gene upstream of the Tet regulatory element. The results showed that cultures of primary hippocampal cells could be transfected with very high efficiency (<70%) by the AdTet-On and AdTet-Off systems. Following transfection with the AdTet-On system no EGFP-fluorescent cells could be detected until doxycycline was added. The AdTet-Off system showed the reverse transcriptional regulation, in that the addition of Tet caused EGFP fluorescence to be abolished.
一种能够高效地将转基因导入神经元并对表达的转基因进行定量控制的转移系统，将极大地促进对神经元基因功能的研究。为了开发这样一个系统，我们将四环素（Tet）响应性的开/关调节元件整合到5型腺病毒（Ad）载体中。通过将增强型绿色荧光蛋白（EGFP）基因置于Tet调节元件上游，来检测转染后转基因表达的调控情况。结果表明，AdTet-On和AdTet-Off系统能够以非常高的效率（<70%）转染原代海马细胞培养物。用AdTet-On系统转染后，在添加强力霉素之前检测不到EGFP荧光细胞。AdTet-Off系统表现出相反的转录调控，即添加Tet会导致EGFP荧光消失。
