Novel Transgenic Mouse Models for L1 Retrotransposons in Development and Disease

L1 逆转录转座子发育和疾病的新型转基因小鼠模型

• 批准号: 8893414
• 负责人: Wenfeng An
• 金额: $ 17.89万
• 依托单位: SOUTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8893414
• 关键词: 5' Untranslated Regions; Accounting; Aging; Animals; Area; Autoimmune Diseases; Autoimmunity; Biological; Biological Assay; Biology; Burn injury; Cell Culture Techniques; Cells; Cellular biology; Characteristics; Cloning; Colorado; DNA Methylation; DNA Transposable Elements; Detection; Development; Disease; Disease model; Electron Microscopy; Elements; Embryonic Development; Ensure; Epigenetic Process; Epitopes; Firefly Luciferases; Foundations; Future; Gametogenesis; Gene Silencing; Gene Transfer Techniques; Generations; Genetic; Genome; Germ Cells; Human; Immunofluorescence Immunologic; Insertional Mutagenesis; Institutes; Inverted Terminal Repeat; L1 Elements; Letters; Long Interspersed Elements; Luciferases; Malignant Neoplasms; Maps; Mediating; Methods; Modeling; Mus; Mutagens; Mutation; National Institute of Child Health and Human Development; National Institute of Drug Abuse; National Institute of Environmental Health Sciences; National Institute of General Medical Sciences; National Institute of Mental Health; National Institute of Neurological Disorders and Stroke; National Institute on Alcohol Abuse and Alcoholism; Neurodegenerative Disorders; Neurodevelopmental Disorder; Open Reading Frames; Pathway interactions; Pennsylvania; Process; Proteins; Pseudogenes; RNA; Regulation; Reporter; Reproductive Biology; Research; Retrotransposition; Retrotransposon; Role; Short Interspersed Nucleotide Elements; Sleeping Beauty; Small RNA; Staging; Testing; Time; Transcriptional Regulation; Transgenes; Transgenic Mice; Transgenic Model; Transgenic Organisms; Universities; Variant; Washington; base; bisulfite; design; design and construction; genetic analysis; human disease; in vivo; male; mouse genome; mouse model; mutant; neurogenesis; novel; novel strategies; piRNA; promoter; public health relevance; tool; transgene expression; tumorigenesis

 DESCRIPTION (provided by applicant): Long interspersed elements type 1 (L1s) are the only active autonomous transposable elements in our genome. L1s replicate themselves but also mobilize short interspersed elements and generate processed pseudogenes. This process is inherently mutagenic and accounts for over 0.1% of sporadic human diseases for which the underlying genetic alteration has been mapped. In addition, elevated L1 activity has been extensively documented during critical developmental stages (e.g., embryogenesis, neurogenesis, gametogenesis, and aging) and various disease processes (e.g., autoimmunity, genotoxic exposure, neurodevelopmental and neurodegenerative disorders, and tumorigenesis). However, the role of L1 retrotransposition during these developmental and disease processes remains poorly defined due to the lack of suitable mouse models. Existing mouse models utilize human L1-based transgenes, which are unlikely to be correctly regulated by endogenous mouse controlling mechanisms, especially when epigenetic mechanisms, such as small RNAs and DNA methylation, are involved. Additionally, the standard transgenesis approach has been used to make these mice and the resulting tandem arrayed transgenes are a target for repeat-induced gene silencing, confounding studies of the transcriptional regulation of these elements. Therefore, the objective of this proposal is to generate novel mouse models for L1 retrotransposons that can be used to interrogate the functional impact of L1 regulation and deregulation in vivo. The new transgenes will feature native mouse L1 elements, including the native promoter and two open reading frames. In addition, both L1 proteins will be epitope tagged to facilitate specific tracking of transgene expression and subcellular localization at the protein level; the transgenes will be marked by recently developed luciferase-based retrotransposition reporter cassettes; and the transgenes will be introduced into the mouse genome via targeted transgenesis to ensure single, dispersed copies. As a proof of principle, the timing of L1 expression and retrotransposition during male germ cell development will be characterized using these new transgenic mouse models. Similar analyses will be performed in a piRNA pathway mutant to evaluate the role of prepachytene piRNAs in controlling insertional mutagenesis by retrotransposons. The proposed project will be performed at one of the world's largest reproductive biology centers, which will allow us to integrate expertise from the fields of transposon biology and germ cell biology. In addition to facilitating our studies, the mouse models derived from this project will have broader applicability, e.g., when combined with other genetically manipulated disease models, they will be useful in elucidating the role of L1 activation under genetic, epigenetic and environmental perturbations, including areas of research sponsored by NCI, NIA, NIAAA, NICHD, NIDA, NIEHS, NIGMS, NIMH and NINDS.

 描述（由申请人提供）：长散布元件 1 型（L1）是我们基因组中唯一活跃的自主转座元件，L1 会自我复制，但也会动员短散布元件并产生加工后的假基因。该过程本质上具有诱变性，占 0.1 以上。已确定其潜在基因改变的散发性人类疾病的百分比 此外，在关键发育阶段（例如，L1 活性升高）已被广泛记录。然而，由于缺乏L1逆转录转座在这些发育和疾病过程中的作用，目前仍不清楚。现有的小鼠模型利用基于人类 L1 的转基因，这些转基因不太可能受到内源性小鼠控制机制的正确调节，特别是当表观遗传机制（例如小 RNA 和 DNA）时。此外，标准的转基因方法已被用于制造这些小鼠，并且由此产生的串联排列的转基因是重复诱导的基因沉默的目标，从而混淆了这些元件的转录调控的研究。该提案的目的是为 L1 逆转录转座子生成新的小鼠模型，该模型可用于研究体内 L1 调节和解除调节的功能影响。新的转基因将以天然小鼠 L1 元件为特征，包括天然启动子和两个开放阅读。此外，两种 L1 蛋白都将被标记，以促进在蛋白质水平上特异性追踪转基因表达和亚细胞定位；转基因将通过最近开发的基于荧光素酶的逆转录报告基因盒进行标记；通过靶向转基因来确保单个、分散的拷贝的小鼠基因组作为原理证明，将使用这些新的转基因小鼠模型来表征雄性生殖细胞发育过程中的 L1 表达和逆转录转座的时间。分析将在 piRNA 途径突变体中进行，以评估前粗线期 piRNA 在通过逆转录转座子控制插入诱变中的作用。拟议的项目将在世界上最大的生殖生物学中心之一进行，这将使我们能够整合以下领域的专业知识。 除了促进我们的研究之外，该项目衍生的小鼠模型还将具有更广泛的适用性，例如，当与其他基因操纵疾病模型结合时，它们将有助于阐明遗传条件下 L1 激活的作用。 、表观遗传和环境扰动，包括 NCI、NIA、NIAAA、NICHD、NIDA、NIEHS、NIGMS、NIMH 和 NINDS 赞助的研究领域。