Generation and characterization of a tamoxifen-inducible lineage tracing tool Cd2-P2A-CreERT2 knock-in mice

Yang Guo ¹ Mengyan Zhu ¹ Zhilan Yu ¹ Qing Li ^2* Yanjuan Chen ^1* Ruling Shen ^1* Ruilin Sun ^2*

• ¹ Shanghai Laboraty Animal Research Center, Shanghai, China
• ² Shanghai Engineering Research Center for model organizations,Shanghai Model Organisms Center, Inc., Shanghai, China

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. Using the CRISPR/Cas9 system, we inserted the CreERT2 transgene expression cassette into the Cd2 gene locus to generate conditional Cre-driver line Cd2-CreERT2 knock-in mice, which drove the expression of CreERT2 by the endogenous Cd2 promoter. By mating the Cd2-CreERT2 strain with a Rosa26-LSL-tdTomato reporter mouse strain which contains a tdTomato expression fragment blocked with a loxP-flanked STOP cassette (LSL) driven by a CAG promoter, a Cd2-CreERT2;Rosa26-LSL-tdTomato reporter strain was obtained to evaluate the expression pattern of CD2 in different cell types. After treatment with tamoxifen, the Cd2-CreERT2 knock-in mice were induced to perform efficient recombination at the loxP 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 more than 30% of cells labelled with tamoxifen-induced tdTomato were CD2 + monocytes, 10% of dendritic cells were tdTomato + /CD2 + cells. Tamoxifen-independent expression of tdTomato occurred in approximately 7% of CD2 + macrophages, but in a negligible (~1%) in CD2 + granulocytes. 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.CD2, CreERT2/loxP system, tdTomato, knock-in, tamoxifen-inducible, conditional gene manipulation Abbreviations Cas CRISPR-associated CRISPR clustered regularly interspaced short palindromic repeats ER estrogen receptor gRNA guide RNA IS immunological synapse DP Double positive SP Single positive