RETROTRANSPOSITION IN L1 TRANSGENIC MICE

L1转座子是一种可移动的DNA元件，已知在包括老鼠和人在内的几个物种中都有活性。众所周知，它们的活动会导致疾病。L1反转录转座子通过复制和粘贴机制移动，并且没有表现出已知的市场整合位点偏好。这些特征使它们成为有用的插入诱变剂，因为它们可以标记它们破坏的基因。L1反转录转座子是一种插入诱变剂，可用于研究肿瘤的进展。通过基因工程，它们可能被设计成以特定组织、特定发育阶段的方式移动。因此，它们可能给癌症遗传学带来革命性的变化。然而，L1转座子在体内的活性还知之甚少。它们移动的频率尚不清楚。反转录转座子在何时何地活跃也不是很清楚。这一建议旨在加深我们对体内L1反转录转座子生物学的理解，并确定它们是否可以作为插入突变剂。为了实现这些目标，我们将演示从整合的染色体位置进行L1逆转录转座。接下来，我们将创建一种用于逆转录转座的可筛选标记分析。这种可筛选的标记分析将使我们能够在不长时间选择抗生素的情况下表征逆转座事件的频率。我们将使用这项测试来评估逆转录转座事件有利的细胞类型和状态。我们希望解决逆转位事件是否受到青睐的问题。我们希望解决逆转录转座事件是否更频繁地发生在转化状态，并促进肿瘤细胞的遗传不稳定性。最后，我们将通过调整我们的L1报告结构来建立L1逆转座的体内模型，以最大限度地增加体内检测的机会。我们将培育我们的L1转基因小鼠，以普遍表达LacZ指示鼠。同时含有标记的L1逆转录转座子和可筛选标记等位基因的小鼠将被用来消除体内逆转录转座的频率，并确定发生逆转录转座事件的组织类型和发育阶段。

L1 transposons are mobile DNA elements which are known to be active in several species including mouse and man. Their activity is known to cause disease. L1 retrotransposons move via a copy and paste mechanism and exhibit no known market integration site preferences. These features make them useful as insertional mutagens because they can tag the genes they disrupt. A insertional mutagens, L1 retrotransposons could be used to study tumor progression. Through genetic engineering they potentially could be designed to move in a tissue-specific, developmental stage- specific fashion. As such, they could revolutionize cancer genetics. However, the activity of L1 transposons in vivo is poorly understood. The frequency with which they move about is not known. It also is not well understood where and when retrotransposons are active. This proposal is designed to further our understanding of the biology of L1 retrotransposons in vivo and establish whether they can serve as insertional mutagens. To achieve these aims we will demonstrate L1 retrotransposition from integrated chromosomal sites. Next we will create a screenable marker assay for retrotransposition. This screenable marker assay will allow us to characterize the frequency of retrotransposition events without prolonged selections in antibiotics. We will use this assay to evaluate the cell types and states in which retrotransposition events are favored. We hope to address whether retrotransposition events are favored. We hope to address whether retrotransposition events occur more frequently in the transformed state and promote genetic instability in tumor cells. Finally, we will establish an in vivo model of L1 retrotransposition by adapting our L1 reporter constructs to maximize the chances of in vivo detection. We will breed our L1 transgenics to ubiquitously expressing lacZ indicator mice. Mice containing both the tagged L1 retrotransposon and the screenable marker allele will be used to eliminate the frequency of retrotransposition in vivo and determine the tissue types and developmental stages in which retrotransposition events occur.