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Poor Concordance of Floxed Sequence Recombination in Single Neural Stem Cells: Implications for Cell Autonomous
Tyler Joseph Dause1, Elizabeth Diana Kirby2,3,4
1Deptartment of Psychology, The Ohio State University, Columbus, OH 43210.
Abstract:
To manipulate target gene function in specific adult cell populations, tamoxifen (TAM)-dependent CreERT2 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 CreERT2-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 CreERT2, 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.
Insights
Tamoxifen-inducible CreERT2 systems are common for gene manipulation. However, using reporter genes to confirm recombination in individual cells is unreliable, as recombination events are not consistently correlated across different genes.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Tamoxifen (TAM)-inducible CreERT2 systems are widely used for inducible, site-specific recombination of loxP-flanked sequences in adult cell populations.
- Stop-floxed fluorescent reporter genes are commonly employed to identify cells undergoing Cre-induced recombination for studying cell-autonomous gene function.
Purpose of the Study:
- To assess the reliability of using Cre-induced recombination of one gene as a predictor for recombination in another gene at the single-cell level.
- To evaluate the concordance of recombination events across different floxed sequences in adult hippocampal neural stem and progenitor cells (NSPCs).
Main Methods:
- Utilized probabilistic predictions within a generic experimental framework.
- Employed a mouse model featuring two distinct stop-floxed reporters and a Nestin promoter-driven CreERT2.
- Analyzed recombination events at the single-cell level in adult hippocampal NSPCs.
Main Results:
- Found that recombination of one gene was a poor predictor of recombination in another gene within individual cells.
- Observed low concordance in floxed sequence recombination across different genes in adult hippocampal NSPCs.
- Demonstrated variability in Cre-mediated recombination efficiency at the single-cell level.
Conclusions:
- The use of stop-floxed reporters to infer cell-autonomous gene function based on Cre-induced recombination may not be universally reliable.
- Poor concordance in recombination suggests potential for false conclusions when relying solely on reporter gene expression.
- Researchers should exercise caution and consider alternative validation methods when investigating cell-autonomous gene function using these systems.
