Poor concordance of floxed sequence recombination in single neural stem cells: Implications for cell autonomous studies

Abstract

To manipulate target gene function in specific adult cell populations, tamoxifendependent 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. Using two separate stopfloxed reporters plus a Nestin promoter-driven CreER T2 , we found that, in individual cells, expression of one reporter was a poor predictor of expression of the other. These findings imply that use of stop-floxed reporters to investigate cell autonomous gene function is likely to lead to false conclusions because recombination in separate genes shows poor concordance in individual cells.

Neural stem and progenitor cells support and protect adult hippocampal function via vascular endothelial growth factor secretion

“…1A). NestinCreER T2 mice drive LoxP recombination with high selectivity in NSPCs when exposed to the synthetic estrogen tamoxifen (TAM) 16,18,19 . We confirmed this selectivity of recombination in NestinCreER T2 mice crossed with stop-floxed EYFP reporter mice, finding that 97% of EYFP+ cells in the DG were NSPCs and less than 1% were astrocytes 3d after TAM (Supplementary Fig.…”

“…Immunolabeling was performed similar to our previous work 18,56 . After perfusion, brains were harvested and fixed 24h in 4% paraformaldehyde in 0.1 M phosphate buffer followed by equilibration in 30% sucrose in PBS, both at 4°C.…”