A FACS-based screen for retroviral silencing: a tool for detecting trans-sensing in mammalian cells

逆转录病毒是一种RNA病毒，它感染细胞，然后整合到被感染细胞的基因组中。这一特性已被用于通过替换有缺陷的基因来治疗遗传疾病：替换的基因可以被纳入逆转录病毒，转运到细胞中，并成为细胞的一部分。S遗传物质。然而，逆转录病毒在整合后一段时间内往往受到宿主细胞的抑制，使其遗传信息变得沉默。导致逆转录病毒沉默的因素尚不清楚，但很明显，这个问题是基于逆转录病毒的（也许是所有的）基因治疗的主要障碍。在各种各样的生物中，包括植物、苍蝇和酵母，已经发现一个基因的多个拷贝可以相互沉默，这种现象被称为共抑制。一些报告表明，这可能也发生在哺乳动物身上。我们已经启动了一项逆转录病毒沉默的研究，使用一种产生绿色荧光的标记蛋白作为替代基因的模型。我们发现该基因通常在整合后立即或随着时间的推移而沉默，但可以通过去甲基化DNA或改变染色体结构的药物重新激活。我们现在建议通过对人类T细胞中逆转录病毒沉默的频率进行系统分析来扩展这项工作，然后开发一个系统来检测和分析逆转录病毒的共抑制。这项工作将依赖于荧光激活细胞分选（FACS）的能力来检测大量细胞中的罕见事件，并恢复这些罕见细胞。我们还将测试打破逆转录病毒沉默状态的方法。这些实验可能会产生有利于广泛的基因替代努力的信息。由于哺乳动物基因组中的内源性元素可能受到这种机制的调节，因此共抑制的论证将特别有趣。

Retroviruses are RNA viruses that infect cells and then become integrated into the genome of the infected cell. This property has been exploited in attempts to cure genetic diseases by replacing the defective gene: the replacement can be incorporated into the retrovirus, transported into a cell, and become part of the cell?s genetic material. However, retroviruses are frequently suppressed by the host cell some time after integration, so that their genetic information becomes silent. The factors that cause silencing of retroviruses are not well understood, but it is clear that this problem is a major impediment to retrovirus-based (and perhaps all) gene therapies. In a wide variety of organisms, including plants, flies, and yeast, it has been found that multiple copies of a gene can silence each other, a phenomenon termed cosuppression. Some reports suggest that this might happen in mammals as well. We have initiated a study of retroviral silencing, using a marker protein that produces green fluorescence as a model for the replaced gene. We find that the gene is usually silenced after integration, immediately or over time, but can be reactivated by drugs that demethylate DNA or alter chromosomal structure. We now propose to extend this work by doing a systematic analysis of the frequency of retroviral silencing in human T cells, and then to develop a system to detect and analyze cosuppression by retroviruses. This work will rely on the ability of fluorescence-activated cell sorting (FACS) to detect rare events in a large population of cells, and recover those rare cells. We will also test methods of destabilizing the silent state of a retrovirus. These experiments are likely to yield information that will benefit a broad array of gene replacement efforts. A demonstration of cosuppression would be particularly interesting because of the possibility that endogenous elements in the mammalian genome are regulated by this mechanism.