Schematic diagrams of the <i>Lamc2</i> allele and transgenes used in these studies and genotyping.

<p>(A) <i>Lamc2</i> null allele is generated by Cre recombinase, which removes exon 8 and the Neo-TK insert. Primer locations are indicated. (B) The K14-rtTA transgene contains a human keratin 14 (K14) promoter driving the reverse tetracycline transactivator (rtTA) and a SV40 poly A signal sequence. (C) The TetO-HuLamC2 transgene contains seven copies of the tetracycline operator (tetO) with a CMV minimal promoter driving the human laminin γ2 cDNA and a bovine growth hormone polyA signal sequence. The binding of doxycycline (Dox) to the rtTA promotes recruitment and binding to the tetO and activation of the promoter. (D) PCR analysis of genomic tail DNA of the <i>Lamc2</i> allele was performed using primers P1, P2, and P3. The mutant allele was detected with primer pair P1–P3, and the wild-type (WT) allele was detected using primer pair P2–P3. The K14-rtTA transgene was detected using K14- and rtTA-specific primers. The human laminin γ2 transgene was detected using primers specific to human laminin γ2. Mice that were a knockout for the <i>Lamc2</i> allele and carried both the K14-rtTA and the TetO-HuLamC2 transgenes (#9) were “rescued” <i>Lamc2</i> KO mice.</p

Localization of hemidesmosomal components is restored in rescued <i>Lamc2</i> KO mice.

<p>Frozen skin sections of <i>Lamc2</i> KO (A–D), rescued <i>Lamc2</i> KO (E–H), and <i>Lamc2</i> WT (I–L) newborn mice were immunostained for skin hemidesmosomal components plectin (A, E, I), BP180/Col XVII (B, F, J), and integrin chains α6 (C, G, K) and ß4 (D, H, L). The immunostaining pattern for all hemidesmosomal proteins in the <i>Lamc2</i> KO mice appeared discontinuous, whereas the staining patterns in rescued <i>Lamc2</i> KO and <i>Lamc2</i> WT mice appeared more linear.</p

Adult tissues of rescued <i>Lamc2</i> KO mice appear grossly similar to <i>Lamc2</i> WT controls.

<p>Paraffin-embedded tissue sections of adult <i>Lamc2</i> WT (A–H) and rescued <i>Lamc2</i> KO (A'–H') mice were stained with H&E. Despite a lack of laminin γ2 expression, the brain (A, A'), heart (B, B'), intestine (C, C'), kidney (D, D'), liver (E, E'), lung (F, F'), spleen (G, G'), and stomach (H, H') appear grossly similar between the <i>Lamc2</i> WT and rescued <i>Lamc2</i> KO mice.</p

Expression of human laminin γ2 facilitates assembly of hemidesmosomes in rescued <i>Lamc2</i> KO mice.

<p>Transmission electron microscopic images of newborn skin of <i>Lamc2</i> KO (A), rescued <i>Lamc2</i> KO (B), and <i>Lamc2</i> WT (C) mice are shown. Hemidesmosomes of newborn <i>Lamc2</i> KO skin are poorly formed, devoid of lamina densa and anchoring filaments, and containing few anchoring fibrils (A). In contrast, rescued <i>Lamc2</i> KO (B) and <i>Lamc2</i> WT (C) mice had well-organized hemidesmosomes with electron dense plaques, anchoring filaments, anchoring fibrils, and darkened areas of lamina densa abutting the hemidesmosomes (arrows). All images are of the same magnification. Bar represents 500 nm.</p

Human laminin γ2 is expressed by keratinocytes and deposited into the basement membrane.

<p>Skin tissue sections from adult <i>Lamc2</i> WT (A–C) and rescued <i>Lamc2</i> KO mice (D–F) were subjected to in situ hybridization for human laminin γ2 mRNA using a digoxigenin-labeled RNA probe (A and D) or immunostaining for human (B and E) or mouse (C and F) laminin γ2 using species-specific laminin γ2 antibodies and TRITC-conjugated secondary antibodies. Blue staining in panels A and D represents positive hybridization. Sections were counterstained with tartrazine yellow for contrast. The lack of staining in panels A and B show that the absence of human laminin γ2 expression in <i>Lamc2</i> WT mice. The lack of staining in panel F shows the absence of mouse laminin γ2 in the rescued <i>Lamc2</i> KO mice.</p

Expression of human laminin γ2 under the K14 promoter prevented blistering of rescued <i>Lamc2</i> KO mice.

<p>The paws (A, D, G), skin (B, E, H), and mouth (C, F, I) of <i>Lamc2</i> KO (A–C), rescued <i>Lamc2</i> KO (D–F), and <i>Lamc2</i> WT (G–I) newborn mice were examined. Skin blistering was most evident on the paws of <i>Lamc2</i> KO (A), but epidermal detachment (B) and separation of the oral mucosa of the roof palate and tongue (arrows in C) were detected microscopically after H&E staining. Blistering was not observed in the rescued <i>Lamc2</i> KO (D–F) or <i>Lamc2</i> WT (G–I) mice.</p

Human laminin γ2 colocalizes with mouse laminin α3 and ß3 chains in rescued <i>Lamc2</i> KO skin.

<p>Frozen skin tissue sections from adult rescued <i>Lamc2</i> KO mice were subjected to immunofluorescence staining for human laminin γ2 (A and B), mouse laminin α3 (C), and mouse laminin ß3 (D) using species-specific anti-laminin γ2 antibodies. Merged images are shown (E and F). Yellow color in panels E and F indicates colocalization.</p

Human laminin γ2 transgene expression is restricted in the rescued <i>Lamc2</i> KO mice.

<p>Whole mount E18 rescued <i>Lamc2</i> KO embryonic tissue sections were stained with hematoxylin and eosin (H&E) (A) or for human laminin γ2 using an anti-human laminin γ2 antibody followed by TRITC-conjugated antibody (B and D). Slides were mounted with mounting media containing DAPI to allow visualization of nuclei (C and E). Human laminin γ2 was only detected in the mouth (B) and skin (D).</p

Alterations in Lm-332 expression do not alter skin differentiation.

<p>Frozen skin sections of <i>Lamc2</i> KO (A–C), rescued <i>Lamc2</i> KO (D–F), and <i>Lamc2</i> WT (G–I) newborn mice were immunostained for skin differentiation markers loricrin (A, D, G), K10 (B, E, H), and K14 (C, F, I). No significant differences were detected in the staining patterns of these skin differentiation markers in <i>Lamc2</i> KO, the rescued <i>Lamc2</i> KO, and <i>Lamc2</i> WT mice. The epidermis of each of these mice displayed loricrin in the granular layer, K10 in the spinous layer, and K14 in the basal layer.</p