The Development Of The Humanized Darc Transgenic Mouse

Abstract

The Duffy Antigen Receptor for Chemokines (DARC) is an atypical chemokine receptor which binds CXC and CC chemokines with no measurable downstream signaling. Erythrocyte DARC serves as a chemokine sink and reservoir. DARC on endothelial cells transports chemokines thus promoting transendothelial leukocyte migration. Two common polymorphisms define the Duffy blood group, FYA and FYB, whilst the third polymorphism which is widely prevalent in West Africa, FYB(ES), abolishes erythrocyte expression of DARC. DARC is a portal of entry for plasmodium vivax suggesting that FYB(ES) was selected due to it conferring resistance to infection. The impact of the FYB(ES) polymorphism extends to a genetic association with the benign ethnic neutropenia. In order to study the mechanisms of this association we have developed a murine transgenic models of the Fy(a-b-) phenotype as encoded by FYB(ES), and Fy(a-b+) phenotype as encoded by FYB. Cloned genes were introduced by pronuclear micro-injection into fertilized oocytes of murine-DARC knockouts. The resulting embryos were transferred into pseudopregnant mice and offspring screened for transgene insertion by PCR genotyping. Gene expression in founder animals was measured by qPCR, demonstrating expression in lung, kidney, liver, spleen, skin and cerebellum. Protein expression was confirmed by immunofluorescence on frozen sections in spleen and skin in a distribution identical to human. Erythrocyte phenotyping by FACS using anti-Fy3 and anti-Fy6 monoclonal antibodies demonstrated the lack of DARC expression in mice with FYB(ES) and confirmed expression on FYB transgenic erythrocytes. We show that murine chemokines bind to human DARC and we plan to use this model to investigate the impact of FYB(ES) on in vivo chemokine functions and equilibria. These models will prove useful in the dissection of the mechanisms of ethnic neutropenia and improve the understanding in the role of DARC in chemokine homeostasis. Disclosures:Chaganti:Roche: Membership on an entity's Board of Directors or advisory committees, Receipt of travel grant Other.