Inherited Junctional Epidermolysis Bullosa in the German Pointer: Establishment of a Large Animal Model Annabelle Capt, Flavia Spirito, Eric Guaguere,

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Inherited Junctional Epidermolysis Bullosa in the German Pointer: Establishment of a Large Animal Model Annabelle Capt, Flavia Spirito, Eric Guaguere, Anne Spadafora, Jean-Paul Ortonne, Guerrino Meneguzzi Journal of Investigative Dermatology Volume 124, Issue 3, Pages 530-535 (March 2005) DOI: 10.1111/j.0022-202X.2004.23584.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Reduced expression of the laminin α3 chain in dog junctional epidermolysis bullosa (JEB). (A) Immunohistochemistry analysis of frozen sections of skin biopsies obtained from a JEB dog (a, c, e, g) and a healthy unrelated control (b, d, f, h) using pAb SE85 (a, b) (Vidal et al, 1995), β3B (c, d) (Matsui et al, 1995), SE144 (e, f) (Vailly et al, 1994) to human laminin α3, β3, and γ2 chains, respectively, and mAb 233 to collagen type XVII (BP180; g, h) (Nishizawa et al, 1993). In the affected dog, immunoreactivity of the laminin α3, β3, and γ2 chains was decreased. Scale bars 50 μm. (B) Northern blot analysis of total RNA purified from primary cultures of wild-type (wt) and JEB (JEB) dog keratinocytes. RNA was successively hybridized with 32P-labeled cDNA probes for the α3, β3, and γ2 chains, and the GAPDH probe (G). The intens ity of the signal for the α3 chain mRNA transcripts appears reduced in JEB keratinocytes. Journal of Investigative Dermatology 2005 124, 530-535DOI: (10.1111/j.0022-202X.2004.23584.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Identification of mutation 4818+207ins 6.5 kb. (A) RT-PCR amplification of total mRNA purified from wild-type (wt) and junctional epidermolysis bullosa (JEB) dog keratinocytes (lanes 1 and 2, respectively), and skin biopsies (lanes 3 and 4, respectively), generated a 659 nucleotides (nt) slow-migrating band (lanes 2 and 4) in the JEB dogs in addition to the wt 432 base pair (bp) cDNA. (B) Nucleotide sequencing of the 659 nt PCR amplification product detected a 227 bp insertion between exons 35 and 36. (C) PCR amplification of wt (lane 1) and JEB (lane 2) intron 35 of lama3 yielded amplification products of 114 bp and 6.5 kb, respectively. M: DNA molecular weight marker. (D) Genomic structure of intron 35 of the morbid lama3 with the endogenous DNA insertion (thick bar). The abnormal α3 mRNA is produced from the aberrant α3 pre-mRNA by activation of the strong cryptic acceptor (A1) and donor (D2) splice sites, whereas production of wild-type α3 mRNA is rescued by the canonical D1 and A2 splice sites. (E) Mendelian inheritance of the morbid lama3 allele in the nuclear family of JEB dogs. The affected (black-filled symbols), wt (empty symbols), and heterozygous healthy carrier (half-filled symbols) dogs are shown. Dots designate dogs not genotyped. The four probands are denoted numerically (1→4), whereas successive ancestral generations are indicated by a Roman numeral. Journal of Investigative Dermatology 2005 124, 530-535DOI: (10.1111/j.0022-202X.2004.23584.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Mutation 4818+207ins6.5 kb hampers keratinocyte proliferation. (A) The junctional epidermolysis bullosa (JEB) keratinocytes secrete reduced amounts of laminin 5. Culture media of wild-type (wt) (lanes 1, 3, and 5) and JEB primary keratinocytes (lanes 2, 4, and 6) were labeled for 16 h with 35S-methionine and 35S-cysteine and immunoprecipitated using either polyclonal antibodies (pAb) SE85 (lanes 1 and 2), pAb β3B (lanes 3 and 4), or pAb SE144 (lanes 4 and 5). Samples were fractionated by electrophoresis under reducing conditions in a 6% polyacrylamide gel. Migration of the α3 (165 kDa), β3 (140 kDa), unprocessed (γ2nc, 155 kDa), and processed γ2 (γ2c, 105 kDa) chains is indicated on the left. Molecular weight markers are indicated on the right. (B) JEB keratinocytes display reduced colony-forming efficiency in culture. JEB or wt dog keratinocytes (5 × 102) were plated and stained 12 d later using rhodamine B. The JEB cells essentially generated abortive colonies. Journal of Investigative Dermatology 2005 124, 530-535DOI: (10.1111/j.0022-202X.2004.23584.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions