# FOXO1 re-expression with a dual-recombinase allele rescues class switching in germinal center B cells

## TL;DR

Scientists developed a new genetic tool to control gene expression in B cells, which helped restore normal immune function when a key gene was missing.

## Contribution

A dual-recombinase system enables sequential gene knockout and re-expression in germinal center B cells.

## Key findings

- Time-controlled FOXO1 re-expression rescues GC compartmentalization defects.
- The system enables efficient and specific gene manipulation in B cells.
- Class switch recombination is functionally restored in FOXO1-deficient cells.

## Abstract

Farré Díaz et al. describe a broadly applicable system for sequential targeted mutagenesis using two recombinases sequentially activated from a single locus. As a proof-of-concept experiment, the time-controlled re-expression of FOXO1 rescued the defects associated with FOXO1-deficient germinal center B cells, including class switch recombination, providing new insights into FOXO1 biology via a dual-recombinase tool.

Modeling complex (patho)physiological processes by sequential targeted mutagenesis in mice is limited by the lack of precision of cellular targeting and complex breeding strategies. We present a new Cre/DreERT2 dual-recombinase germinal center B cell (GCBC)–specific strain, with co-expression of the recombinases from a single allele. This enables highly efficient Cre-mediated FOXO1 knockout in GCBCs in vivo, followed by time-controlled, efficient Dre-mediated FOXO1 re-expression in the same cells, leading to functional rescue of GC compartmentalization and class switch recombination. The present approach can be easily adapted to other cellular contexts.