title: Characterization of tetracycline-inducible bitransgenic Krt12rtTA/+/tet-O-LacZ mice.
abstract: OBJECTIVE
  To prepare binary transgenic mouse lines that overexpress reporter genes in a corneal-epithelium-specific manner when induced by doxycycline.
  
  
  METHODS
  A gene-targeting construct containing an internal ribosomal entry site-reverse tetracycline transcription activator (IRES-rtTA) cassette was inserted into the Krt12 allele (keratin 12 gene) to produce a knock-in Krt12(rtTA/+) mouse line through gene-targeting techniques. The Krt12(rtTA/+) knock-in mice were bred with tet-O-LacZ reporter mice to obtain Krt12(rtTA/+)/tet-O-LacZ bitransgenic mice. The expression of the LacZ gene was induced in bitransgenic mice by administration of doxycycline in the drinking water and chow.
  
  
  RESULTS
  Administration of doxycycline induced a 15-fold increase of beta-galactosidase activity in the cornea of adult bitransgenic mice (Krt12(rtTA/+)/tet-O-lacZ). Administration of doxycycline either to single transgenic Krt12(rtTA/+) or tet-O-LacZ mice as a control did not induce overexpression of LacZ as it did in the bitransgenic mice. The induction of beta-galactosidase enzyme activity by doxycycline in bitransgenic mice took place in 24 hours and reached a plateau by 2 days. Histochemical analysis also showed that beta-galactosidase induction was limited to the corneal epithelium of bitransgenic mice fed doxycycline. The increased beta-galactosidase activity in corneal epithelium caused by doxycycline returned to basal levels in 4 weeks after the antibiotics were omitted from the diet.
  
  
  CONCLUSIONS
  A binary mouse model has been successfully established that conditionally overexpresses reporter genes in corneal epithelium. This mouse model will be useful in elucidating signaling pathways of various growth factors and cytokines and gene functions in the maintenance of homeostasis and pathogenesis in the adult mouse cornea.
authors.affiliation: Department of Ophthahlmology, University of Cincinnati, Ohio 45267, USA.
authors.forename: Tai-Ichiro
authors.initials: T
authors.lastname: Chikama
authors.forename: Yasuhito
authors.initials: Y
authors.lastname: Hayashi
authors.forename: Chia-Yang
authors.initials: CY
authors.lastname: Liu
authors.forename: Noriko
authors.initials: N
authors.lastname: Terai
authors.forename: Kazuto
authors.initials: K
authors.lastname: Terai
authors.forename: Candace W-C
authors.initials: CW
authors.lastname: Kao
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.forename: Miyuki
authors.initials: M
authors.lastname: Hayashi
authors.forename: Teruo
authors.initials: T
authors.lastname: Nishida
authors.forename: Philip
authors.initials: P
authors.lastname: Sanford
authors.forename: Tom
authors.initials: T
authors.lastname: Doestchman
authors.forename: Winston W-Y
authors.initials: WW
authors.lastname: Kao
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion; D015153:Blotting, Western; D004318:Doxycycline / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression / Q000502:physiology*; D018390:Gene Targeting; D017930:Genes, Reporter / Q000502:physiology*; D005822:Genetic Vectors; D005838:Genotype; D053551:Keratin-12; D007633:Keratins / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D012333:RNA, Messenger / Q000378:metabolism; D013752:Tetracycline; D015533:Transcriptional Activation; D019076:Transgenes; D001616:beta-Galactosidase / Q000096:biosynthesis
chemical_list: C507495:KRT12 protein, mouse; D053551:Keratin-12; D012333:RNA, Messenger; D007633:Keratins; D001616:beta-Galactosidase; D013752:Tetracycline; D004318:Doxycycline