RETROTRANSPOSITION IN L1 TRANSGENIC MICE

L1 转基因小鼠的逆转录转座

• 批准号: 6633548
• 负责人: ELINE Tjetske LUNING PRAK
• 金额: $ 13.66万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-10 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6633548
• 关键词: HeLa cells; Retroviridae; beta galactosidase; genetic markers; genetic transcription; genetically modified animals; green fluorescent proteins; laboratory mouse; mutagens; nucleic acid sequence; plasmids; transfection; transfection /expression vector; transposon /insertion element

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.

L1转座子是一种移动的DNA元件，已知其在包括小鼠和人在内的多种物种中具有活性。L1反转录转座子通过复制和粘贴机制移动，并且没有已知的市场整合位点偏好。这些特征使它们可以作为插入诱变剂，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突变剂，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清楚。该建议旨在进一步了解L1反转录转座子在体内的生物学，并确定它们是否可以作为插入诱变剂。为了实现这些目标，我们将证明从整合的染色体位点L1逆转录转座。接下来，我们将建立一个可筛选的标记物测定逆转录转座。这种可筛选的标记物测定将使我们能够表征逆转录转座事件的频率，而无需延长抗生素的选择。我们将使用该试验来评价逆转录转座事件有利的细胞类型和状态。我们希望解决逆转录转座事件是否有利。我们希望解决逆转录转座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，我们将建立一个体内模型的L1逆转录转座，通过调整我们的L1报告结构，以最大限度地提高体内检测的机会。我们将培育我们的L1转基因无处不在地表达lacZ指示小鼠。含有标记的L1反转录转座子和可筛选标记等位基因的小鼠将用于消除体内反转录转座的频率，并确定发生反转录转座事件的组织类型和发育阶段。