study of mice genome dynamics in the context of Trex1 KO.. Trex

We present genomic sequencing data from two experiments, which might be relevant for people interested in genome dynamics in the context of Trex-1 KO mutant, especially people interested in repetitive elements: > a) we have successfully bred two parallel lines of Trex1-/- mice and we have the sequence of one individual from each line after inbreeding (one brother-sister) of 7 and 11 generations respectively, A7 and B11. > b) we also sequenced Trex1-/- ku80-/- animals from a double heterozygous cross. We sequenced the 4 genotypes from the cross (one generation only because the double homozygote is sterile): double heterozygote (T+K+), single heterozygote for one gene and double for the other (T-K+ and T+K-), and double homozygote (T-K-). There are arguments from comparative genome analysis during evolution that some organisms loose their introns. All current models of intron loss involve reverse transcription products generated from spliced mRNAs that can serve as a template for gene conversion-type recombination events that can eliminate introns at the genomic level. In Trex1-/- mice, these reverse transcription products accumulate in such large quantities that they activate cytoplasmic surveillance mechanisms that induce an interferon response. The central hypothesis of this work is that the accumulation of these reverse transcription products in the cells will have important genomic consequences, such as intron loss. In order to maintain healthy Trex1 homozygous KO mice, we balanced the Trex1 mutation with a deficiency in the Zap70 gene that prevents T cell activation. The Trex1-/- Zap70-/- double KO mice can reproduce in the laboratory. Our project was to sequence the genome of these mice after about 10 generations of inbreeding. The fertility of these inbred families is low and the litters are small, but we were able to continue our experiment of propagating a genome in a Trex1 deficient context. At the seventh generation, we sacrificed an individual in order to prepare genomic DNA and sequence the genome (Illumina, 125 bp, paired) and also of an individual from another independent family at generation 11. Our research must have been good because we identified new copies lacking introns, but which corresponded to pseudogenes inserted after reverse transcription compared to the mouse reference genome we were using (despite the numerous backcrosses on C57Bl6, there remain some regions of line 129) . However, we have shown that they were in fact pre-existing the Trex1 KO in the animals we used To increase the chance of observing an intron loss event by increasing the frequency of homologous recombination, we also constructed a double KO line: Trex1-/- and Ku80-/-, a gene required for non-homologous recombination (NHEJ). The Ku80- homozygous individuals are viable but not fertile, this time we sequenced mices of 4 possible genotypes from a double heterozygous Trex+/-, Ku80+/- cross: double heterozygous Trex+/-, Ku80+/-, double homozygous Trex-/-, Ku80-/- as well as the respective single homozygotes Trex-/-, Ku+/- and Trex+/-, Ku80-/-. Unfortunately for our hypothesis, no intron deletion events were detected after genotype analysis of the double homozygous individual vs. the other 3. You are welcome to use the data. Clement Mettling ([e-mail]), Aubin Thomas ([e-mail]) and Georges Lutfalla ([e-mail])

本数据集包含两项实验的基因组测序数据，可供关注Trex-1敲除（Trex-1 KO）突变背景下基因组动态、尤其是重复序列研究的科研人员参考。
a) 我们成功培育了两株平行的Trex1敲除（Trex1-/-）小鼠品系，分别经过7代和11代兄妹近交后，对每一品系的1只个体进行了全基因组测序，对应品系分别命名为A7和B11。
b) 我们还对双杂合杂交产生的Trex1-/- ku80-/- 个体进行了测序。本次杂交共产生4种基因型（仅进行单代测序，因双纯合子不育）：双杂合子（"T+K+"）、单基因杂合且另一基因纯合的个体（"T-K+"和"T+K-"），以及双纯合子（"T-K-"）。

进化过程中的比较基因组学研究表明，部分生物会丢失内含子。目前所有已报道的内含子丢失模型均涉及：从剪接成熟mRNA生成的逆转录产物可作为模板，通过基因转换型重组事件在基因组层面消除内含子。在Trex1-/-小鼠体内，这类逆转录产物会大量积累，激活胞质监控机制并诱发干扰素应答。本研究的核心假说为：细胞内这类逆转录产物的积累会对基因组产生重要影响，例如内含子丢失。

为维持健康的Trex1纯合敲除小鼠，我们通过引入Zap70基因缺陷（该缺陷可阻断T细胞活化）来平衡Trex1突变。Trex1-/- Zap70-/- 双敲除小鼠可在实验室环境中繁殖。本项目的目标是对经过约10代近交的这类小鼠进行全基因组测序。这些近交繁育的小鼠生育率较低、产仔量偏小，但我们仍得以在Trex1缺陷背景下完成了基因组传代实验。在第7代时，我们处死1只个体以提取基因组DNA并进行测序（采用Illumina测序平台，125bp双端测序模式），同时对另一独立品系第11代的1只个体进行了相同测序。本研究取得了有价值的结果：我们鉴定出了一批缺失内含子的新拷贝序列，但相较于我们使用的小鼠参考基因组（尽管在C57Bl6背景上进行了多次回交，仍残留部分129品系的基因组区域），这些序列实为逆转录后插入的假基因（pseudogenes）。不过我们后续证实，这些假基因实际上在实验所用动物的Trex1敲除模型建立前就已存在。

为提升同源重组频率，从而增加观察到内含子丢失事件的概率，我们还构建了双敲除品系：Trex1-/- 与Ku80-/-（Ku80是非同源重组（non-homologous recombination, NHEJ）通路的必需基因）。Ku80纯合敲除个体可存活但不育。本次我们对双杂合子Trex+/-、Ku80+/- 杂交产生的4种基因型小鼠进行了全基因组测序：双杂合子Trex+/-、Ku80+/-、双纯合子Trex-/-、Ku80-/-，以及对应的单纯合子Trex-/-, Ku+/- 和Trex+/-, Ku80-/-。遗憾的是，与其余3种基因型个体相比，双纯合子个体的基因组分析未检测到内含子缺失事件。

欢迎各位科研人员使用本数据集。
作者：克莱门特·梅特林（Clement Mettling，[e-mail]）、奥班·托马斯（Aubin Thomas，[e-mail]）及乔治·卢法拉（Georges Lutfalla，[e-mail]）