Fig 1.
Characterization of Col7a1del8/del8 rat phenotype.
A) Kaplan-Meier survivorship curve (n = 12) of Col7a1del8/del8 pups. Median survival = 3.5 days, mean survival = 5.7 days. B-C) Prone size comparison between B) Col7a1flNeo/Neo and WT hypomorphic mouse pups [9] and C) Col7a1del8/del8 and WT rat pups. D) Paw of Col7a1del8/del8 pup presenting with pseudosyndactyly, and E) WT littermate. F) Col7a1del8/del8 pup in supine position displaying both intact and burst nonhemorrhagic blistering. Arrows indicate locations of cutaneous blistering at various stages of healing. G) Right lateral recumbency of Col7a1del8/del8 pup with blistering of ear. H) Col7a1del8/del8 pup with cutaneous blistering to posterior. Arrows indicate the affected areas on the anatomical right-rear limb, tail and belly. I) WT rat pup littermate.
Fig 2.
Immunofluorescence and H&E staining of RDEB rat skin, demonstrating respective aberrant collagen type VII formation and blistering within the DEJ.
A-B) Rat WT and Col7a1del8/del8 skin costained with LSbio collagen VII antibody (red) and DAPI (blue) at 40× magnification; scale bars = 50 μm. C-F) H&E staining of skin from one-day-old WT and Col7a1del8/del8 rat pups at 40× magnification; scale bars = 100 μm. Double arrows indicate areas of hyperkeratosis observed. Areas of mild multifocal epidermal hyperplasia are noted with single arrows. Subepidermal clefting was observed in Col7a1del8/del8 samples (*). Arrowheads point to areas of mild multifocal hypercellularity of superficial dermis.
Fig 3.
Transmission electron microscopy of WT and Col7a1del8/del8 skin.
Low magnification demonstrating A) intact WT skin and B) full-thickness separation (*) of epithelium (Ep) from dermis (Derm) in Col7a1del8/del8 skin. Distinct arching and looping anchoring fibrils (af, arrows) extend into the dermis and entrap banded collagen fibrils in C) WT skin but are absent in D) Col7a1del8/del8 skin. The plane of separation in Col7a1del8/del8 skin is deep to the lamina densa (LD), which is thinner and less dense than in WT. Fibrous material (fib, arrows) is associated with the LD in D) Col7a1del8/del8 skin. E) Immuno-gold labeling (arrows) for type VII collagen is strongly positive in WT skin at the lamina densa (LD), with most intense labeling below hemidesmosomes (HD) where anchoring fibrils (af, arrowheads) are concentrated. F) Type VII collagen labeling was absent in Col7a1del8/del8 skin. The sub-epithelial LD is poorly defined in intact regions due to accumulation of fibrous material (*). Scale bars: A,B = 100 microns; C-F = 500 nm.
Fig 4.
Characterization of the CRISPR-Cas9 induced mutagenic region.
A) PCR-fluorescent fragment analysis genotyping assay of founder animals run in duplicate. Double peaks at approximately 343bp and 351bp in length correspond to heterozygous animals, while single peaks at 351bp correspond to WT. B) Sanger sequencing chromatograms and alignment of WT and Col7a1del8/del8 rats. Displayed sequencing results from C7 Rev primer (5’-AGGCAAGATTAGGAAGGACTTGGGG-3’). WT and Col7a1del8/del8 chromatograms had minimal noise and evenly spaced peaks throughout. An 8bp deletion (red box) was observed in Col7a1del8/del8 results within exon 1. The protospacer adjacent motif (PAM) is noted (black box) and the gRNA target sequence is highlighted yellow. C) Predicted amino acid sequence of exon 1 revealing frameshift mutation and potential introduction of premature stop codon (*). Differences in amino acid sequence are noted within the red box.