- Function research of genes
- Function research of microglial cells and other Cx3cr1-expressing immune cells
Homozygous mice for the targeted mutation are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. In this strain, iCre/ERT2 fusion protein and EGFP expression is under the control of Cx3cr1 promoter. When crossed with a strain containing a loxP-site flanked sequence of interest, Cre-mediated recombination will result in deletion of the floxed sequence in the offspring.
Gene editing strategy
A targeting vector was designed to replace 390bp of exon 2 of the Cx3cr1 gene with a iCre/ERT2 (improved cre recombinase fulsed to estrogen receptor 1) coding sequence, followed by a porcine teschovirus-1 2A (P2A) and a enhanced green fluorescent protein (EGFP). The targeting vector were injected into C57BL/6N mouse zygotes. After injection, surviving 2-cell-stage zygotes were transplanted to the KM albino pseudopregnant females. The reslulting Cx3cr1-iCreERT2-EGFP founder mice were bred to C57BL/6N mice. These mice were maintained on a C57BL/6N background.
The expression of EGFP and tdTomato in mouse spleen, bone marrow, and brain mononuclear macrophages was detected by flow cytometry, and Cx3cr1 (Mut /+) was found after induction of Tamoxifen.The majority of macrophages in the brain of tdTomato (Mut/+) mice were double positive cells of EGFP and tdTomato, while the mononuclear macrophages in spleen and bone marrow were single positive cells of EGFP. The results were consistent with the results in the study, indicating that B-Cx3cr1-iCreERT2-EGFP mice were designed and expressed successfully.
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