Identification of the cornea-specific keratin 12 promoter by in vivo particle-mediated gene transfer.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Shiraishi
A
A
Department of Ophthalmology, University of Cincinnati, Ohio 45267-0527, USA.
Converse
R L
RL
Liu
C Y
CY
Zhou
F
F
Kao
C W
CW
Kao
W W
WW
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001483:Base Sequence; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D003228:Conjunctiva / Q000378:metabolism; D004247:DNA / Q000032:analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019573:Epithelium, Corneal / Q000378:metabolism*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D018398:Homeodomain Proteins; D006801:Humans; D007633:Keratins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D011401:Promoter Regions, Genetic; D011817:Rabbits; D012097:Repressor Proteins; D012867:Skin / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D014157:Transcription Factors; D007633:Keratins; D004247:DNA; D001616:beta-Galactosidase