RETROTRANSPOSITION IN L1 TRANSGENIC MICE

L1转座子是一种可移动的DNA元件，已知在包括小鼠和人类在内的几种物种中都有活性。已知它们的活动会导致疾病。L1逆转录转座子通过复制和粘贴机制移动，不表现出已知的市场整合位点偏好。这些特征使它们作为插入诱变剂很有用，因为它们可以标记它们所破坏的基因。插入诱变剂，L1逆转录转座子可用于研究肿瘤进展。通过基因工程，它们有可能被设计成以组织特异性、发育阶段特异性的方式移动。因此，它们可以彻底改变癌症遗传学。然而，L1转座子在体内的活性尚不清楚。他们移动的频率是未知的。反转录转座子在何时何地活跃也不是很清楚。本研究旨在进一步了解L1逆转录转座子在体内的生物学特性，并确定它们是否可以作为插入诱变剂。为了达到这些目的，我们将展示整合染色体位点的L1反转录转位。接下来，我们将创建一个可筛选的标记检测逆转录转位。这种可筛选的标记试验将使我们能够表征逆转录转位事件的频率，而不需要长期选择抗生素。我们将使用这个试验来评估细胞类型和状态，其中逆转录转位事件是有利的。我们希望解决逆行转位事件是否有利。我们希望解决逆转录转位事件是否在转化状态下更频繁地发生，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逆转录转位的体内模型，以最大限度地提高体内检测的机会。我们将把我们的L1转基因基因培育到无处不在表达lacZ的小鼠身上。同时含有标记L1反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反转录转座子的频率，并确定发生反转录转座子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.