# Normal macrophage signaling and gene expression in Rosa26 Cas9-expressing mice

## TL;DR

This study confirms that macrophages from commonly used Rosa26-Cas9 mice function normally in key signaling pathways.

## Contribution

Demonstrates normal macrophage signaling in widely used Cas9-FZ mice under standard conditions.

## Key findings

- Cas9-FZ mice show no defects in macrophage signaling pathways like TLR3 and TLR4.
- RNA sequencing and immunoblotting confirm normal gene expression and signaling.
- Differences in culture techniques may explain prior conflicting results.

## Abstract

Cas9-expression from the Rosa26 “safe harbor” locus are widely used for gene manipulation and Crispr-based screening. Recently, experimental evidence suggested that macrophages isolated from Rosa26-Cas9 mice may have signaling differences compared to control mice in terms of TRIF signaling downstream of TLR3 and TLR4. As we frequently use the Rosa26-Cas9 mice made by Feng Zhang (Cas9-FZ, Jackson Laboratory stock No. 026179), arguably the strain with the widest distribution and utilization, we were motivated to test macrophage signaling in these mice under our conventional conditions. We used different macrophage polarization and signaling conditions combined with RNA sequencing and measurement of TLR signaling by immunoblotting. Our results suggest that the Cas9-FZ mice bear no obvious defects in any commonly used macrophage signaling pathway. We document the differences in our macrophage culture techniques compared to Raychowdhury et al., which may aid in how individual laboratories use Cas9-expressing macrophages, especially for focused or genome-wide screening.