Figure 1

Inducible model of p27^Kip1 inactivation in adult mice. (A) The p27 coding region was conditionally targeted by generating mice harboring LoxP sites at the p27 locus (p27^L+) and expressing a tamoxifen-regulated Cre recombinase under the control of the chimeric chicken beta-actin promoter, CAGG::CreER™. (B) Experimental design: Tamoxifen was administered by single daily intraperitoneal injections (i.p.) for 5 consecutive days in 2 month-old mice. For proliferation analysis, BrdU was injected once daily for three consecutive days starting 8 days after the first tamoxifen injection. All timepoints were calibrated to the start of tamoxifen exposure. (C) Western blots for p27 and the marker of glial reactivity GFAP. Each lane contains 15 μg of protein from individual retinas. The blot was cut at ~35 kDa: portions with the lower and higher molecular weight bands were stained for p27 and GFAP, respectively. (D and E) Immunostaining against p27 and the Müller glial marker Glutamine Synthetase (GLUL). Micrographs of control (p27^L-/L+) and experimental animals (p27^L-/L-) six weeks after the start of tamoxifen injections. Asterisks indicate blood vessels. Arrowheads denote Müller glia that failed to undergo p27 inactivation. (F) Inactivation efficiency was measured by comparing the number of p27⁺ cells colocalizing with the glial nuclear marker SOX9. Data are expressed as the mean ± SD. Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; RGC, retinal ganglion cell layer; p27, cyclin-dependent kinase inhibitor CDKN1B; GFAP, glial fibrillary acidic protein; GLUL, glutamine synthetase; SOX9, SRY-box containing gene 9; and DAPI, 4', 6-diamidino-2-phenylindole.