Poor Concordance of Floxed Sequence Recombination in Single Neural Stem Cells: Implications for Cell Autonomous Studies
Poor Concordance of Floxed Sequence Recombination in Single Neural Stem Cells: Implications for Cell Autonomous Studies
复制标题
DOI：
10.1523/eneuro.0470-19.2020
复制
发表时间：
2020-03-01
期刊：
影响因子：
3.4
通讯作者：
Kirby, Elizabeth Diana
中科院分区：
文献类型：
--
作者：
Dause, Tyler Joseph;Kirby, Elizabeth Diana
为了操纵特定成体细胞群中的靶基因功能，他莫昔芬 (TAM) 依赖性 CreER(T2) 被广泛用于驱动 loxP 侧翼序列的诱导型、位点特异性重组。在细胞自主靶基因功能的研究中，通常的做法是将这些 CreER(T2)-lox 系统与普遍表达的 stop-floxed 荧光报告基因结合起来，以识别可能正在经历靶基因重组的单个细胞。在这里，我们研究了在成体海马神经干和祖细胞 (NSPC) 的单细胞水平上使用 Cre 诱导的一个基因重组来预测另一个基因重组的可靠性。使用通用实验范例中的概率预测，以及具有两个独立的 stop-floxed 报告基因和 Nestin 启动子驱动的 CreER(T2) 的小鼠模型，我们发现，在单个细胞中，一个基因的重组对于另一个基因的重组来说是一个很差的预测因子。基因间 floxed 序列重组的这种较差的一致性表明，使用 stop-floxed 报告基因来研究细胞自主基因功能可能并不普遍可靠，并且可能导致错误的结论。
To manipulate target gene function in specific adult cell populations, tamoxifen (TAM)-dependent CreER(T2) is widely used to drive inducible, site-specific recombination of loxP flanked sequences. In studies of cell autonomous target gene function, it is common practice to combine these CreER(T2)-lox systems with a ubiquitously expressed stop-floxed fluorescent reporter gene to identify single cells supposedly undergoing target gene recombination. Here, we studied the reliability of using Cre-induced recombination of one gene to predict recombination in another gene at the single-cell level in adult hippocampal neural stem and progenitor cells (NSPCs). Using both probabilistic predictions in a generic experimental paradigm, as well as a mouse model with two separate stop-floxed reporters plus a Nestin promoter-driven CreER(T2), we found that, in individual cells, recombination of one gene was a poor predictor of recombination in another. This poor concordance in floxed sequence recombination across genes suggests that use of stop-floxed reporters to investigate cell autonomous gene function may not be universally reliable and could lead to false conclusions.