Characterization of the Rabbit Neonatal Fc Receptor (FcRn) and Analyzing the Immunophenotype of the Transgenic Rabbits That Overexpresses FcRn

Abstract

The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes an active role in phagocytosis, and delivers antigen for presentation. We have previously shown that overexpression of FcRn in transgenic mice significantly improves the humoral immune response. Because rabbits are an important source of polyclonal and monoclonal antibodies, adaptation of our FcRn overexpression technology in this species would bring significant advantages. We cloned the full length cDNA of the rabbit FcRn alpha-chain and found that it is similar to its orthologous analyzed so far. The rabbit FcRn - IgG contact residues are highly conserved, and based on this we predicted pH dependent interaction, which we confirmed by analyzing the pH dependent binding of FcRn to rabbit IgG using yolk sac lysates of rabbit fetuses by Western blot. Using immunohistochemistry, we detected strong FcRn staining in the endodermal cells of the rabbit yolk sac membrane, while the placental trophoblast cells and amnion showed no FcRn staining. Then, using BAC transgenesis we generated transgenic rabbits carrying and overexpressing a 110 kb rabbit genomic fragment encoding the FcRn. These transgenic rabbits – having one extra copy of the FcRn when hemizygous and two extra copies when homozygous - showed improved IgG protection and an augmented humoral immune response when immunized with a variety of different antigens. Our results in these transgenic rabbits demonstrate an increased immune response, similar to what we described in mice, indicating that FcRn overexpression brings significant advantages for the production of polyclonal and monoclonal antibodies.

FcRn Overexpression in Transgenic Mice Results in Augmented APC Activity and Robust Immune Response with Increased Diversity of Induced Antibodies

“…Our previous analyses have shown that the bFcRn Tg mice offer major advantages for hybridoma production and could serve as important tools for the development of new therapeutic mAbs [4]. In addition, we have recently generated Tg rabbits that overexpress the rabbit FcRn and observed similarly improved IgG protection and enhanced humoral immune response as described for bFcRn Tg mice [5].…”

Immunoglobulin Transport and Immunoglobulin Receptors

“…The range of animals in which FcRn orthologs have been identified includes humans, mice, rats, rabbits (Catunda Lemos et al, 2012), cattle (Laegreid et al, 2002), sheep (Mayer et al, 2002), Egyptian water buffalo (Sayed-Ahmed et al, 2010), camels (Kacskovics et al, 2006), pigs (Schnulle and Hurley, 2003), and possums (Catunda Lemos et al, 2012). Homology between the human and rat FcRn is 68% (West and Bjorkman, 2000), whereas rat FcRn is 91% homologous to mouse FcRn (Ahouse et al, 1993).…”

“…Bovine FcRn is 77% homologous to human FcRn, as is rabbit FcRn (Kacskovics et al, 2000). Across species, the least homology is seen in the cytoplasmic tail of FcRn, where the length can vary by more than 10 amino acids (Catunda Lemos et al, 2012). As discussed below, both variations in cytoplasmic tail features and differences in glycosylation patterns contribute to the minor discrepancies in IgG binding and function observed for FcRn across species.…”

Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons

“…In addition to this low transgenesis rate, the integration site and copy number of a plasmidbased transgene is uncontrolled, therefore unwanted ectopic expression or transgene silencing might occur. Transgenesis by artificial chromosomes (YAC, BAC) ensures improved expression patterns, because those large-size vectors include most if not all the regulatory regions and the expression domains of the gene of interest (GOI) 2,11 . However, microinjection of transgenes cloned into artificial chromosomes is technically demanding due to their large size and fragility, and the integrity of the chromosomally integrated transgene should be strictly controlled.…”

“…The rabbit is the primary source of polyclonal antibodies, and recent transgenic rabbit models are capable of a significantly increased level of antibody production 1,2 . Transgenic rabbits are also used as bioreactors for the production of pharmaceutical proteins.…”