long kidney function relies on the complement of nephrons generated during mammalian development from a mesenchymal nephron progenitor cell population. Low nephron endowment confers increased susceptibility to CKD. Reduced nephron numbers in the popular Six2TGC transgenic mouse line may be due to disruption of a regulatory gene at the integration site and/or ectopic expression of a gene(s) contained within the transgene. Methods Targeted locus amplification was performed to identify the integration site of the Six2TGC transgene. Genome-wide chromatin conformation capture (Hi-C) datasets were generated from nephron progenitor cells isolated from the Six2TGC+/tg mice, the Cited1CreERT2/+ control mice, and the Six2TGC+/tg; Tsc1+/Flox mice that exhibited restored nephron number compared with Six2TGC+/tg mice. Modified transgenic mice lacking the C-terminal domain of Six3 were used to evaluate the mechanism of nephron number reduction in the Six2TGC+/tg mouse line. Results Targeted locus amplification revealed integration of the Six2TGC transgene within an intron of Cntnap5a on chr1, and Hi-C analysis mapped the precise integration of Six2TGC and Cited1CreERT2 transgenes to chr1 and chr14, respectively. No changes in topology, accessibility, or expression were observed within the 50-megabase region centered on Cntnap5a in Six2TGC+/tg mice compared with control mice. By contrast, we identified an aberrant regulatory interaction between a Six2 distal enhancer and the Six3 promoter contained within the transgene. Increasing the Six2TGCtg to Six2 locus ratio or removing one Six2 allele in Six2TGC+/tg mice caused severe renal hypoplasia. Furthermore, clustered regularly interspaced short palindromic repeats disruption of Six3 within the transgene (Six2TGC∆Six3CT) restored nephron endowment to wild-type levels and abolished the stoichiometric effect. Conclusions These findings broadly demonstrate the utility of Hi-C data in mapping transgene integration sites and architecture. Data from genetic and biochemical studies together suggest that in Six2TGC kidneys, SIX3 interferes with SIX2 function in nephron progenitor cell renewal through its C-terminal domain....

中文翻译：

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Six2-TGCtg 小鼠中肾单位的减少是由于转基因中异常的增强子-启动子相互作用导致 Six3 错误表达

长期肾功能依赖于哺乳动物发育过程中由间充质肾单位祖细胞群产生的肾单位的补充。肾单位禀赋较低导致 CKD 的易感性增加。流行的 Six2TGC 转基因小鼠系中肾单位数量的减少可能是由于整合位点调节基因的破坏和/或转基因中包含的基因的异位表达。方法进行靶向基因座扩增以确定 Six2TGC 转基因的整合位点。全基因组染色质构象捕获 (Hi-C) 数据集是从 Six2TGC+/tg 小鼠、Cited1CreERT2/+ 对照小鼠和 Six2TGC+/tg 小鼠中分离的肾单位祖细胞生成的；与 Six2TGC+/tg 小鼠相比，Tsc1+/Flox 小鼠表现出肾单位数量恢复。使用缺乏 Six3 C 端结构域的修饰转基因小鼠来评估 Six2TGC+/tg 小鼠系中肾单位数量减少的机制。结果 靶向位点扩增揭示了 Six2TGC 转基因在 chr1 上 Cntnap5a 内含子内的整合，Hi-C 分析将 Six2TGC 和 Cited1CreERT2 转基因分别精确整合到 chr1 和 chr14。与对照小鼠相比，Six2TGC+/tg 小鼠中以 Cntnap5a 为中心的 50 兆碱基区域内没有观察到拓扑结构、可及性或表达的变化。相比之下，我们发现转基因中包含的 Six2 远端增强子和 Six3 启动子之间存在异常的调控相互作用。在 Six2TGC+/tg 小鼠中增加 Six2TGCtg 与 Six2 基因座的比例或去除一个 Six2 等位基因会导致严重的肾发育不全。此外，转基因内的 Six3 (Six2TGCΔSix3CT) 内的成簇规则间隔的短回文重复序列破坏将肾单位禀赋恢复到野生型水平并消除了化学计量效应。结论 这些发现广泛证明了 Hi-C 数据在绘制转基因整合位点和架构方面的效用。遗传和生化研究的数据表明，在 Six2TGC 肾脏中，SIX3 通过其 C 末端结构域干扰 SIX2 在肾单位祖细胞更新中的功能......