Deptartment of Psychology, The Ohio State University, Columbus, OH 43210.
Deptartment of Psychology, The Ohio State University, Columbus, OH 43210
eNeuro. 2020 Mar 20;7(2). doi: 10.1523/ENEURO.0470-19.2020. Print 2020 Mar/Apr.
To manipulate target gene function in specific adult cell populations, tamoxifen (TAM)-dependent CreER 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-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, 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.
为了在特定的成年细胞群体中操纵靶基因功能，他莫昔芬（TAM）依赖性 CreER 被广泛用于驱动loxP 侧翼序列的诱导、特异性重组。在研究细胞自主靶基因功能时，通常将这些 CreER-lox 系统与广泛表达的停止-floxed 荧光报告基因结合使用，以鉴定据称正在发生靶基因重组的单个细胞。在这里，我们研究了在成年海马神经干细胞和祖细胞（NSPCs）中，使用 Cre 诱导的一个基因重组来预测另一个基因在单细胞水平上重组的可靠性。我们使用通用实验范式中的概率预测，以及具有两个独立的停止-floxed 报告基因加 Nestin 启动子驱动的 CreER 的小鼠模型，发现单个细胞中一个基因的重组是另一个基因重组的不良预测指标。这种跨基因 floxed 序列重组的不良一致性表明，使用停止-floxed 报告基因来研究细胞自主基因功能可能不是普遍可靠的，并且可能导致错误的结论。
