Generation and characterization of a tamoxifen-inducible lineage tracing tool knock-in mice.

INTRODUCTION

The new targeted gene editing technologies, such as the CRISPR/Cas system, enable researchers to insert or delete genes at targeted loci efficiently. The Cre-loxp recombination system is widely used to activate or inactivate genes with high spatial and temporal specificity.

METHODS

Using the CRISPR/Cas9 system, we inserted the transgene expression cassette into the gene locus to generate conditional Cre-driver line knock-in mice, which drove the expression of CreERT2 by the endogenous promoter. By mating the strain with a reporter mouse strain which contains a tdTomato expression fragment blocked with a loxP-flanked STOP cassette (LSL) driven by a CAG promoter, a reporter strain was obtained to evaluate the expression pattern of CD2 in different cell types.

RESULTS

After treatment with tamoxifen, the knock-in mice were induced to perform efficient recombination at the site following CreERT2 activation and cause the expression of tdTomato fluorescence. The tdTomato and CD2 were expressed in the T cells of peripheral blood, spleen and mesenteric lymph nodes, whereas detected in a low proportion in the B cells. While about 20% of cells labeled with tamoxifen-induced tdTomato were CD2 monocytes in peripheral blood, 10% of dendritic cells were tdTomato/CD2 cells. Tamoxifen-independent expression of tdTomato occurred in approximately 3% of CD2 macrophages, but in negligible (~0.5%) in CD2 granulocytes.

DISCUSSION

This work supplied a new transgenic mouse as a valuable tool for lineage tracing in CD2-expressing cells, for conditional mutant studies of immune modulatory effects in a time-dependent manner, and analysis of the potential therapeutic effect of CD2-targeting biologics.