Fig 1.
Schematic diagram for generation of the targeted TGFBI allele.
Wild type allele, targeting construct, targeted allele, and Neo removed allele are shown. The human TGFBI cDNA sequence containing R124H mutation was knocked-in to the mouse Tgfbi exon 1. The detail of targeting is described in Materials and Methods.
Fig 2.
Generation of TGFBIR124H Tg mice.
(A) Southern blot analysis of targeted ES cells. The black bars on 5′ and 3′ outside of the targeting region in panel A indicate the locations serving as probes for genotyping by Southern blot. 15.4-kb and 4.6-kb of genomic DNA fragment is produced by Ndel digestion from wild type allele and knocked-in allele respectively, and detected by 5’ probe. 12.3-kb and 9.8-kb of genomic DNA fragment is produced by HindIII digestion from wild type allele and knocked-in allele respectively, and detected by 3’ probe. (B) Removal of Neo cassette was confirmed by genotyping PCR. The Primer pair produces 1.3-kb and 2.8-kb PCR products in absence and presence of Neo cassette, respectively. No PCR product is produced from wild type allele. (C) Representative PCR genotyping result of progenies (F12) obtained by mating TGFBI+/KI heterozygous mice with C57BL/6J mouse. Usual genotyping PCR were performed with primer pairs for knocked-in allele and wild-type allele, which give rise to 1385-bp and 787-bp of PCR products, respectively. (D) Human TGFBI mRNA is found in the TGFBIKI/KI homozygous mice (F12) and mouse Tgfbi mRNA is not. RT-PCR analysis was performed using human TGFBI gene and mouse Tgfbi gene specific primer pairs. Total RNA samples prepared from cultured mouse cells, wild-type mouse skin, human corneal cells, and cultured human cells were used as positive controls respectively. The primer pairs for human GAPDH gene and mouse Gapdh gene were used as reaction control as well. (E) TGFBI protein is found in TGFBIKI/KI mouse cornea by immunohistochemistry. The sections of corneas prepared from TGFBIKI/KI homozygous mouse, heterozygous mouse and wild type mouse were stained with anti-TGFBi polyclonal antibody and visualized with DAB. Dense staining is observed beneath the epithelium in KI/ KI mouse and +/KI mouse (arrow head). Scale bar = 100 um in E.
Table 1.
Oligonucleotide sequences for primer pairs used in PCR genotyping.
Table 2.
Oligonucleotide sequences for primer pairs used in RT-PCR.
Fig 3.
Granular and lattice deposits without corneal edema and neovascularization were observed in the center of the cornea in homozygous (52 weeks, female, A, B, 78 weeks, male, C, D) and heterozygous (52 weeks, female, E, F, 94 weeks female, G, H) mice. Wild type mice had clear corneas (I).
Fig 4.
In homozygotes, HE staining did not show signs of inflammation in the cornea (A) as well as wild type mice (B), while Masson trichrome staining showed red deposits in the anterior cornea (C) in contrast to wild type mice (D). Congo red stating showed red deposits (arrow head) in the anterior cornea (E) and birefringence of the area was observed (G) in contrast to wild type mice (F, H). Subepithelial stroma was also stained with thioflavin T (*) in homozygotes (I) in contrast to wild type mice (J). Positive control: Human renal amyloidosis (K). Non-specific staining of cell nuclei is observed in both samples and positive control. Immunohistochemical examination with LC3 showed subepitheilaicl stromal staining (*) in homozygotes (L) in contrast to wild type mice (M). Examination with electronic microscopy revealed subepithelial deposits (*) in heterozygotes (N) in contrast to wild type mice (O). Autophagosomes (arrow head) were observed around the deposits. Scale bar = 100 um in A—J, 1 um in K, L.
Table 3.
Demographic features.
Table 4.
Incidence of corneal opacity.