title: Generation of Cre transgenic mice with postnatal RPE-specific ocular expression.
abstract: OBJECTIVE
  To generate and characterize a constitutively active, RPE-specific, cre-expressing transgenic mouse line. This line can be used to create RPE-specific knockouts by crossing with mice harboring loxP-flanked (floxed) genes.
  
  
  METHODS
  A transgene construct was assembled with the BEST1 promoter driving cre expression. Transgenic mice were generated on a C57BL/6 background. Cre expression was assessed by immunofluorescence and Western blot analysis. Cre enzymatic activity was tested by crossing to three lines with floxed DNA regions and detecting deletion of the intervening sequences or through histochemical detection of lacZ activity. Potential cre-mediated toxicity was assessed by retinal histology up to 24 months of age and by electroretinography.
  
  
  RESULTS
  The BEST1-cre line with expression in the highest percentage of RPE cells displayed a patchy mosaic expression pattern, with 50% to 90% of RPE cells expressing cre. In mice outcrossed to a mixed B6/129 background, expression was consistently found in 90% of RPE cells. Within the eye, only the RPE cells were immunoreactive with an anti-cre antibody. Maximum cre expression quantified by Western blot analysis occurred at P28. Crosses with three lines containing floxed sequences revealed RPE-specific cre activity in the eye and extraocular expression limited to the testes. Histology and electroretinography showed no cre-mediated RPE toxicity.
  
  
  CONCLUSIONS
  This BEST1-cre transgenic line enables generation of RPE-specific knockout mice. The mosaic expression pattern provides an internal control; the non-cre-expressing RPE cells continue to express the floxed genes. These mice should facilitate study of the multifunctional RPE and the generation of mouse models of human retinal disease.
authors.affiliation: F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Jared
authors.initials: J
authors.lastname: Iacovelli
authors.forename: Chen
authors.initials: C
authors.lastname: Zhao
authors.forename: Natalie
authors.initials: N
authors.lastname: Wolkow
authors.forename: Peter
authors.initials: P
authors.lastname: Veldman
authors.forename: Kandace
authors.initials: K
authors.lastname: Gollomp
authors.forename: Pallavi
authors.initials: P
authors.lastname: Ojha
authors.forename: Nina
authors.initials: N
authors.lastname: Lukinova
authors.forename: Ayala
authors.initials: A
authors.lastname: King
authors.forename: Leonard
authors.initials: L
authors.lastname: Feiner
authors.forename: Noriko
authors.initials: N
authors.lastname: Esumi
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Zack
authors.forename: Eric A
authors.initials: EA
authors.lastname: Pierce
authors.forename: Douglas
authors.initials: D
authors.lastname: Vollrath
authors.forename: Joshua L
authors.initials: JL
authors.lastname: Dunaief
mesh_terms: D000818:Animals; D000075002:Bestrophins; D015153:Blotting, Western; D004596:Electroretinography; D005136:Eye Proteins / Q000235:genetics; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D019426:Integrases / Q000235:genetics*; D007473:Ion Channels / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D055213:Retinal Pigment Epithelium / Q000201:enzymology*
chemical_list: C491856:Best1 protein, mouse; D000075002:Bestrophins; D005136:Eye Proteins; D007473:Ion Channels; C045073:Cre recombinase; D019426:Integrases