Oddly enough, when staining wild-type human skin vis–vis wild-type murine skin collagen VII we consistently noticed a high reactivity to human collagen VII but low reactivity to murine collagen VII with antisera from all three rabbits (Supplemental Fig. on wild-type mice for preclinical therapy studies. These approaches are challenged by collagen VII expression by the murine host. Thus, the ability to selectively visualize human and murine collagen VII would be a substantial advantage. Here, we describe TMPA a novel resource toward this TMPA end. By immunization with homologous peptides we generated rabbit polyclonal antibodies that recognize either human or murine collagen VII. Testing on additional species, including rat, sheep, dog, and pig, combined sequence alignment and peptide competition binding assays enabled identification of the major antisera recognizing epitopes. The species-specificity was maintained after denaturation and the antibodies allowed us to simultaneously, specifically visualize human and murine collagen VII gene therapy by transplantation of gene-corrected epidermal grafts for JEB and DEB, it has emerged that the curative effect is less sustained for DEB [6]. This is likely a reflection of key differences in the biology and functions of the two proteins at fault [7], and highlights the need to continue exploring alternative or complementary therapeutic avenues for DEB. The development of therapies for DEB is challenged by early neonatal lethality of complete collagen VII deficient mice [8]. A collagen VII hypomorphic mouse model that presents most manifestations of severe DEB has been developed [9] and has been widely used by us and others for therapy development and to better understand DEB pathobiology [[10], [11], [12], [13], [14], [15]]. However, for therapies aiming to restore collagen VII abundance the remaining residual expression of wild-type collagen VII can obscure the results. Complementary to genetic mouse models, studies on topical curative therapies, but also other lines of therapies, have frequently used grafting of human skin equivalents or similar onto immunodeficient mice [[16], [17], [18]]. Nevertheless, the evaluation of collagen VII abundance and deposition in the grafts can be influenced by collagen VII expressed by the wild-type host [19]. In this context C and for all other preclinical studies introducing human collagen VII in murine hosts C tools to specifically detect human and murine collagen VII would be valuable. We and others have shown that the mouse monoclonal collagen VII antibody LH 7: 2 [20,21], which is widely used for diagnostics to detect human collagen VII, recognizes human but not murine collagen VII [12,19]. However, commonly used hosts for human skin and cell grafting such as SCID or athymic nude mice [[16], [17], [18]] are leaky TMPA or maintain production of immunoglobulins which distorts staining with mouse monoclonal antibodies. The generation of murine and human specific collagen VII antibodies is challenged by the rather homogenous 84% sequence identity of human and murine collagen VII over the entire polypeptides. In an effort to generate a potent collagen VII body we previously cloned a 246-amino acid peptide of the central non-collagenous (NC)-1 domain of human collagen VII reported to harbor the epitope of LH 7:2 [22] and raised a rabbit polyclonal antiserum against this peptide [12]. The resulting antiserum was highly potent in western blotting, ELISA and immunofluorescence, unexpectedly C because of the large size of the peptide used for immunization and the homology of human and murine collagen VII C we noted low reactivity to murine collagen VII [12]. Here, we re-raised the antibody in multiple rabbits and observed persistent high human collagen VII and low murine collagen VII reactivity of the generated antibodies. This indicated intrinsic properties of the peptide used for immunization favoring human but not murine collagen VII recognition. Based on this observation and prediction suggesting divergence of the most antigenic sequence between human and murine collagen VII, we raised antibodies against the corresponding murine peptide. The resulting antibodies reacted strongly with murine but not human collagen VII in cell and tissue staining and western blotting. Together these antibodies thus constitute a unique, useful tool to distinguish human and murine collagen VII which we practically showed by injection of human collagen VII into wild-type mouse skin. Results and discussion The high amino acid sequence conservation between mouse and human collagen VII makes generation of polyclonal antibodies that TMPA show Th preference for reactivity with one of the species challenging (Fig. 1A). The most sequence-divergent part of human and murine collagen VII is in the C-terminal NC-2 domain, in which an additional stretch of 11 extra amino acids is found in the murine collagen VII alpha 1 chain. However, since.
Oddly enough, when staining wild-type human skin vis–vis wild-type murine skin collagen VII we consistently noticed a high reactivity to human collagen VII but low reactivity to murine collagen VII with antisera from all three rabbits (Supplemental Fig