A MOUSE MODEL FOR STUDYING LINE-1 REGULATION IN VIVO

Simon James Newkirk

One of the most illuminating discoveries from genome sequencing is the abundance of transposable elements in mammalian genomes. About half of the human genome is composed of various classes of transposable elements. The single largest group, by mass, is the Long Interspersed Element type 1 (LINE−1, or L1), a copy-paste retrotransposon. L1s are unique among transposable elements, in humans, in that they still possess all of the machinery necessary to mobilize themselves, and are actively retrotransposing in the human genome. In order to survive, L1s are under constant pressure to evade host suppression and to pass new insertions to future generations. Current data suggest that L1 insertions occur primarily in the male germline or early in embryogenesis. However, given the abundant and repetitive nature of endogenous L1s, it is very difficult to identify accurately when and where new insertions take place. The objective of this dissertation is to develop an L1 transgenic mouse model that represents the endogenous L1 activity, in order to facilitate the investigation of L1 regulation, retrotransposition, and its impact on the genome. Here we describe the characterization of a hyperactive L1 transgene that is driven by an endogenous mouse L1 promoter, and show that it mimics endogenous regulation. Our data show comparative levels of hypomethylation during fetal reprogramming and subsequent de novo methylation, similar to the endogenous L1s. Quantification of insertions from this transgene displays a highly variable frequency across tissues, suggesting the involvement of multiple rate- limiting steps. The utility of our transgenic model is further illustrated by introducing the transgene into the piRNA deficient, Mov10l1 mouse knockout background. For the first time, we reported abundant retrotransposition in piRNA-deficient testes. Further detailed analysis showed L1 overexpression and massive retrotransposition, specifically, in meiotic spermatocytes. We believe this transgene will serve as an invaluable tool not only in advancing our fundamental knowledge about L1 biology but also in providing critical insights into the role of L1 activities in many physiopathological processes, such as during cancer progression and aging.

A MOUSE MODEL FOR STUDYING LINE-1 REGULATION IN VIVO

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