Preclinical double-targeted hGITR/Foxp3 reporter mouse model
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Design of the hGITR/Foxp3 mouse
The humanized GITR/Foxp3 model was generated by intercrossing hGITR and Foxp3-IRES-mRFP (FIR) reporter mice.
The hGITR is developed by Knockin at the mouse GITR locus, and expresses a chimeric GITR with a human extracellular and murine intracellular domain. hGITR is regulated by the endogenous mouse promoter. The FIR's bicistronic reporter expressing a red fluorescent protein (RFP) has been knocked into the endogenous Foxp3 locus and faithfully mirrors Foxp3 expression.
hGITR/Foxp3 features
- The GITR extracellular domain is entirely humanized
- Physiological regulation and expression pattern of the human GITR
- Fully functional mouse immune system
- Lack of expression of the murine GITR, thus avoiding cross-reactivity
- Physiological expression of murine Foxp3
- RFP reporter faithfully mirrors Foxp3 expression
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Validation
hGITR expression pattern in hGITR/Foxp3 mice recapitulates mGITR expression in wild-type mice
Expression of human and mouse GITR on αCD3/αCD28-stimulated splenocytes (5 days), analyzed by flow cytometry on Tregs and conventional CD4+ (CD4conv) T cells.
RFP expression mirrors Foxp3 protein expression
Expression of mRFP on non-permeabilized (A) or permeabilized (B) freshly isolated splenocytes (CD3+CD4+) from hGITR/Foxp3 mice. mRFP+ cells were sorted and permeabilized to allow Foxp3 detection (C).
Suppressive function of Treg is preserved
Isolated CD4+CD25- Teff cells were labeled with CTV and cultured with various concentrations of isolated RFP+ Treg cells in presence of antigen-presenting cells treated with mitomycin and αCD3 for 4 days. Proliferation was assessed by flow cytometry (CTV+ cells) on viable cells. Results were analysed by Student t test *p<0.001.
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hGITR/Foxp3
Preclinical double-targeted hGITR/Foxp3 reporter mouse model
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