Kao W W, Liu C Y, Converse R L, Shiraishi A, Kao C W, Ishizaki M, Doetschman T, Duffy J
Department of Opthalmology, University of Cincinnan, Ohio 45267-0527, USA.
Invest Ophthalmol Vis Sci. 1996 Dec;37(13):2572-84.
Expression of the K3-K12 keratin pair characterizes the corneal epithelial differentiation. To elucidate the role of keratin 12 in the maintenance of corneal epithelium integrity, the authors bred mice deficient in keratin 12 by gene-targeting techniques.
One allele of murine Krt1.12 gene was ablated in the embryonic stem cell line, E14.1, by homologous recombination with a DNA construct in which the DNA element between intron 2 and exon 8 of the keratin 12 gene was replaced by a neo-gene. The homologous recombinant embryonic stem cells were injected to mouse blastocysts, and germ lines of chimeras were obtained. The corneas of heterozygous and homozygous mice were characterized by clinical observations using stereomicroscopy, histology with light and electron microscopy, Western immunoblot analysis, immunohistochemistry, in situ hybridization, and Northern hybridization.
The heterozygous mice (+/-) one allele of the Krt1.12 gene appear normal and do not develop any clinical manifestations (e.g., corneal epithelial defects). Homozygous mice (-/-) develop normally and suffer mild corneal epithelial erosion. Their corneal epithelia are fragile and can be removed by gentle rubbing of the eyes or brushing with a Microsponge. The corneal epithelium of the homozygote (-/-) does not express keratin 12 as judged by immunohistochemistry, Western immunoblot analysis with epitope-specific anti-keratin 12 antibodies, Northern hybridization with 32P-labeled keratin 12 cDNA, and in situ hybridization with an anti-sense keratin 12 riboprobe. Light and electron microscopy revealed subtle abnormalities in the corneal epithelia of -/- mice (i.e., a decrease in number of cell layers) and cytolysis of superficial cells, but the number of hemidesmosomes and desmosomes are normal in basal and suprabasal cells. The number of keratin intermediate filaments in basal and suprabasal corneal epithelial cells in -/- mice decreases, and they appear as dense bundles. This morphology is similar to that of keratin intermediate filaments in epidermal epithelial, cells but differs from that of normal corneal epithelial cells in which the keratins form fine filamentous networks. The superficial epithelial cells are devoid of keratin intermediate filaments and often detach from the corneal surface of -/- mice.
The presence of cornea-specific K3-K12 keratin pairs is essential for the maintenance of corneal epithelium integrity.
K3 - K12角蛋白对的表达是角膜上皮分化的特征。为阐明角蛋白12在维持角膜上皮完整性中的作用，作者通过基因靶向技术培育了角蛋白12缺陷的小鼠。
在胚胎干细胞系E14.1中，通过与一种DNA构建体进行同源重组，将小鼠Krt1.12基因的一个等位基因切除，该构建体中角蛋白12基因内含子2和外显子8之间的DNA元件被新霉素基因取代。将同源重组胚胎干细胞注射到小鼠囊胚中，获得嵌合体的种系。通过体视显微镜进行临床观察、光镜和电镜组织学检查、Western免疫印迹分析、免疫组织化学、原位杂交和Northern杂交，对角蛋白12杂合子和纯合子小鼠的角膜进行特征分析。
Krt1.12基因一个等位基因的杂合子小鼠（+/-）外观正常，未出现任何临床表现（如角膜上皮缺损）。纯合子小鼠（-/-）发育正常，但患有轻度角膜上皮糜烂。它们的角膜上皮很脆弱，通过轻轻揉眼或用微海绵擦拭即可去除。通过免疫组织化学、用表位特异性抗角蛋白12抗体进行的Western免疫印迹分析、用32P标记的角蛋白12 cDNA进行的Northern杂交以及用反义角蛋白12核糖探针进行的原位杂交判断，纯合子（-/-）的角膜上皮不表达角蛋白12。光镜和电镜显示-/-小鼠角膜上皮有细微异常（即细胞层数减少）以及表层细胞的细胞溶解，但基底细胞和基底上层细胞中的半桥粒和桥粒数量正常。-/-小鼠角膜基底细胞和基底上层细胞中的角蛋白中间丝数量减少，且呈现为致密束状。这种形态与表皮上皮细胞中的角蛋白中间丝相似，但与正常角膜上皮细胞不同，正常角膜上皮细胞中的角蛋白形成精细的丝状网络。表层上皮细胞缺乏角蛋白中间丝，并且经常从-/-小鼠的角膜表面脱落。
角膜特异性K3 - K12角蛋白对的存在对于维持角膜上皮完整性至关重要。
