New vector system for the gene therapy of abdominal cancers.

在时间和定量方面对基因表达的严格控制在基础和临床生物学中都是一个非常宝贵的工具。诱导系统的期望特征如下。(I)该调节器应仅在给予外源性介质时激活，并在去除外源性刺激时终止。(ii)介质应无毒且在口服给药时有活性。(iii)该系统不应激活其他内源性细胞基因。我们收缩的逆转录病毒载体，具有一个自动调节盒，并表达感兴趣的基因，以响应口服多西环素（dox）体内。该盒含有逆转录四环素调节（rtTA）系统（Gossen M.et al.1995）的所有组分、具有内部核糖体进入位点（IRES）的药物选择标记（BSD）和目的基因（GFP）。逆转录病毒长期重复增强子和启动子元件驱动taransactivator（rTetR-VP 16）和BSD的表达，但 ...更多信息 翻译在BSD的3 ′-位点终止。GFP的表达受串联泰特操纵子序列和巨细胞病毒最小启动子的调控，并与dox呈剂量依赖关系。流式细胞仪分析表明，GFP-荧光诱导的逆转录病毒感染的208 F细胞依赖于剂量的两个数量级。经口给予阿霉素可诱导裸鼠腹腔内移植的208 F细胞表达绿色荧光蛋白。因此，这种逆转录病毒载体系统（RTRRV）可以将可控基因快速地传递到培养的细胞和转基因动物中。然而，控制系统在几个人类癌细胞中不起作用。这可能是由于种属依赖性的易感性，以病毒。我们还发现，RTRRV中引入的毒性基因杀死了PA 317逆转录病毒包装细胞，这表明RTRRV仍然存在非诱导条件下基因表达基础水平较高和宿主细胞基因多效性的问题。如果我们能解决上述问题，这种诱导型逆转录病毒载体系统可能为基因治疗应用中特定时间窗内的基因表达调控铺平道路。少

Tight control of gene expression in temporal and quantitativemanner is an invaluable tool in both basic and clinical biology. The desired characteristics of the inducible system are as follows. (I) The regulator should be activated only upon administration of an exogenousmediator, and terminated when the exogenous stimulus is removed. (ii) The mediator should be non toxic and active upon oral administration. (iii) The system should not activate other endogenous cellular genes. We constricted a retroviral vector that has an autoregulatory cassette and express the gene of interest in response to oral administration of doxycycline (dox) invivo. The cassette contains the all components of the reversetetracycline-regulated (rtTA) system (Gossen M.et al. 1995), a drugselectable marker (BSD) with the internal ribosome entry site (IRES) and the gene of interest (GFP). The retroviral long term repeat enhancer and promoter elements driv-expression of the taransactivator (rTetR-VP16) and BSD, but … More the translation was terminated at the 3'-site of BSD. The expression of GFP was controlled under the tandem tet operator sequencesand the cytomegalovirus minimal promoter in a dose-dependent manner to dox. FACS analyses showed that GFP-fluoresence was induced in two-ordermagnitude in the retrovirus-infected 208F cells dependent on the amount of dox. Furthermore oral administration of dox could induce GFP protein in thetransplanted 208F cells in the peritoneal cavity of a nude mouse. Thus, this reverse tetracycline-regulated retroviral vector (RTRRV) system allowseasy delivery of controllable genes to cultured cells and transgenicanimals. However, the control system did not work in several human cancercells. This may be attributed to the species-dependent susceptibility toretorviruses. And we found that the introduction of a toxic gene into RTRRVkilled the PA317 retrovirus packaging cells, suggesting that RTRRV still suffer from problems of higher basal levels of gene expression undernoninduced condition and pleiotropic effects on host cell genes. If we will solve these problems mentions above, this inducible retroviral vector system may pave the way to the controlled gene expression during a particular window of time in gene therapy applications. Less