An Wenfeng, Han Jeffrey S, Wheelan Sarah J, Davis Edward S, Coombes Candice E, Ye Ping, Triplett Christina, Boeke Jef D
High Throughput Biology Center and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, MD 21205, USA.
Proc Natl Acad Sci U S A. 2006 Dec 5;103(49):18662-7. doi: 10.1073/pnas.0605300103. Epub 2006 Nov 21.
Long interspersed element type 1 (L1) retrotransposons are ubiquitous mammalian mobile elements and potential tools for in vivo mutagenesis; however, native L1 elements are relatively inactive in mice when introduced as transgenes. We have previously described a synthetic L1 element, ORFeus, containing two synonymously recoded ORFs relative to mouse L1. It is significantly more active for retrotransposition in cell culture than all native L1 elements tested. To study its activity in vivo, we developed a transgenic mouse model in which ORFeus expression was controlled by a constitutive heterologous promoter, and we established definitive evidence for ORFeus retrotransposition activity both in germ line and somatic tissues. Germ line retrotransposition frequencies resulting in 0.33 insertions per animal are seen among progeny of ORFeus donor element heterozygotes derived from a single founder, representing a >20-fold increase over native L1 elements. We observe somatic transposition events in 100% of the ORFeus donor-containing animals, and an average of 17 different insertions are easily recovered from each animal; modeling suggests that the number of somatic insertions per animal exceeds this number by perhaps several orders of magnitude. Nearly 200 insertions were precisely mapped, and their distribution in the mouse genome appears random relative to transcription units and guanine-cytosine content. The results suggest that ORFeus may be developed into useful tools for in vivo mutagenesis.
长散在重复序列1型（L1）逆转座子是普遍存在的哺乳动物移动元件，也是体内诱变的潜在工具；然而，当作为转基因引入时，天然L1元件在小鼠中相对不活跃。我们之前描述了一种合成L1元件ORFeus，相对于小鼠L1，它包含两个同义重新编码的开放阅读框（ORF）。在细胞培养中，它的逆转座活性比所有测试的天然L1元件都显著更高。为了研究其在体内的活性，我们构建了一种转基因小鼠模型，其中ORFeus的表达由组成型异源启动子控制，并且我们在生殖系和体细胞组织中都建立了ORFeus逆转座活性的确凿证据。在来自单个奠基者的ORFeus供体元件杂合子的后代中，观察到生殖系逆转座频率为每只动物0.33次插入，这比天然L1元件增加了20倍以上。我们在100%含有ORFeus供体的动物中观察到体细胞转座事件，并且平均每只动物很容易检测到17个不同的插入；模型表明，每只动物的体细胞插入数量可能超过这个数字几个数量级。近200个插入被精确映射，并且它们在小鼠基因组中的分布相对于转录单元和鸟嘌呤 - 胞嘧啶含量似乎是随机的。结果表明，ORFeus可能被开发成为体内诱变的有用工具。
