Shiraishi A, Converse R L, Liu C Y, Zhou F, Kao C W, Kao W W
Department of Ophthalmology, University of Cincinnati, Ohio 45267-0527, USA.
Invest Ophthalmol Vis Sci. 1998 Dec;39(13):2554-61.
Keratin 12 (K12) is a cornea epithelial cell-specific intermediate filament component. To provide a better understanding of its expression, it is necessary to identify and characterize the promoter of Krt1.12 gene.
The 2.5-kb DNA 5' to Krt1.12 gene was sequenced. Krt1.12 promoter-beta-gal DNA constructs were prepared and used in vivo to transfect rabbit corneas, conjunctivas, and skin by particle-mediated gene transfer (Gene Gun). In vitro, the DNA constructs were transfected into cultured T-antigen-transformed rabbit corneal epithelial (RCE-T) cells and human fibrosarcoma HT-1080 fibroblasts with lipofectamine. The promoter activity was assessed by measuring beta-gal (beta-galactosidase) activity using histochemical staining with 5-Bromo-4-chloro-3-indolyl-beta-D-galactoside and enzyme assay with o-nitrophenyl beta-D-galactopyranoside.
There are four Pax-6 pair box binding elements found between -910 and -2000 bp 5'-flanking the transcription initiation site of the Krt1.12 gene. None of promoter constricts can be expressed by HT-1080 cells. Cotransfection of Pax-6 cDNA with K12 promoter-beta-gal constructs containing Pax-6 elements results in a fourfold increase of beta-gal activities in RCE-T cells but not HT-1080 fibroblasts. The data of in vivo transfection in the rabbit by Gene Gun indicate that reporter gene constructs containing 0.6-kb and longer DNA fragments 5'-flanking Krt1.12 gene are effectively expressed in corneal, but not conjunctival or epidermal epithelial cells.
The particle-mediated gene transfer is a suitable technique for in vivo delivery of transgenes to corneal epithelial cells. The 2.5-kb DNA fragment 5'-flanking Krt1.12 contains corneal epithelial cell-specific regulatory cis-DNA elements. Pax-6 is a positive transcription factor essential for keratin 12 expression.
角蛋白12（K12）是角膜上皮细胞特异性中间丝成分。为了更好地了解其表达情况，有必要鉴定和表征Krt1.12基因的启动子。
对Krt1.12基因5'端上游2.5kb的DNA进行测序。制备Krt1.12启动子-β-半乳糖苷酶DNA构建体，并通过粒子介导的基因转移（基因枪）在体内转染兔角膜、结膜和皮肤。在体外，用脂质体将DNA构建体转染到培养的T抗原转化兔角膜上皮（RCE-T）细胞和人纤维肉瘤HT-1080成纤维细胞中。使用5-溴-4-氯-3-吲哚基-β-D-半乳糖苷进行组织化学染色并采用邻硝基苯基-β-D-吡喃半乳糖苷进行酶测定，通过测量β-半乳糖苷酶（β-gal）活性来评估启动子活性。
在Krt1.12基因转录起始位点5'侧翼-910至-2000bp之间发现了四个配对盒结合元件。HT-1080细胞不能表达任何启动子构建体。将Pax-6 cDNA与含有Pax-6元件的K12启动子-β-半乳糖苷构建体共转染，导致RCE-T细胞中β-半乳糖苷活性增加四倍，但HT-1080成纤维细胞中未增加。基因枪在兔体内进行转染的数据表明，含有Krt1.12基因5'侧翼0.6kb及更长DNA片段的报告基因构建体在角膜上皮细胞中有效表达，但在结膜或表皮上皮细胞中不表达。
粒子介导的基因转移是一种将转基因体内递送至角膜上皮细胞的合适技术。Krt1.12基因5'侧翼的2.5kb DNA片段包含角膜上皮细胞特异性调控顺式DNA元件。Pax-6是角蛋白12表达所必需的正性转录因子。
