Method for Treating Ocular Cancer

Mouse and Rat Strains

[0038]Rb+ / − mice were obtained from The Jackson Laboratory (Bar Harbor, Me.). The p53Lox / Lox and RbLox / Lox mice were obtained from the National Cancer Institute (Bethesda, Md.). The p107 knockout mice, Chx10-Cre mice, and Pax6-Cre mice are well-known in the art (see Lee et al. (1996) Genes Dev. 10:1621-1632; Rowan & Cepko (2004) Dev. Biol. 271(2):388-402; Davis-Silberman, et al. (2005) Hum. Mol. Genet. 14(15):2265-76, respectively). All mice were crossed to C57Bl / 6 mice purchased from Charles River Laboratories (Wilmington, Mass.). Timed-pregnant Sprague Dawley rats were obtained from Charles River Laboratories. Newborn pups received an intravitreal injection of 1,000 Y79-LUC cells that constitutively expressed firefly luciferase (Laurie, et al. (2005) supra). The detection of retinoblastoma growth and luciferase with the Xenogen system is known in the art (Laurie, et al. (2005) supra).

Antibodies, Immunostaining, BrdU, [3H]Thymidine, and TUNEL

[0039]Immunolabeled retinal sections cut on a vibratome and dissociated retinae (250-500 cells per sample in triplicate) were prepared according to established methods (Mendrysa, et al. (2003) Mol. Cell. Biol. 23:462-72; Laurie, et al. (2005) supra). The following antibodies were used: the anti-MDMX monoclonal antibodies 6B1A and 11F4D, and polyclonal sera p55 and p56 are known in the art (Ramos, et al. (2001) Cancer Res. 61:1839-42) to human MDMX; DO-1, PAb1801 and PAb240 anti-p53 mouse monoclonals to human p53 (Santa Cruz Biotechnology, Santa Cruz, Calif.); Pab 246 anti-p53 mouse monoclonal to mouse p53 (Santa Cruz Biotechnology); anti-Phospho Ser-15 p53 rabbit polyclonal (Cell Signaling Technology, Danvers, Mass.); 10H11.E12 anti-Phospho ATM mouse monoclonal to human ATM (Rockland Immunochemicals, Gilbertsville, Pa.); SMP14 anti-MDM2 mouse monoclonal to human MDM2 SMP14 (GENETEX, San Antonio, Tex.) and 4B2, and anti-MDM2 rabb

Square-Wave Electroporation and FACS

[0040]Retinae were electroporated in vivo at P0 by injecting 0.5 μL CsCl preparation plasmid DNA (5 μg / μL) into the eye. Electroporation consisted of five pulses of 80 V for 50 μsec each separated by 950-μsec recovery periods. As a control, retinae were electroporated with a plasmid lacking Cre. For explant cultures, the DNA was purified and resuspended in HBSS (1 μg / μL). Electroporation consisted of five pulses of 25 V for 50 μsec each separated by 950-μsec recovery periods. For FACS purification of electroporated cells, retinae were dissociated according to established methods (Dyer & Cepko (2001a) supra; Dyer & Cepko (2001b) supra), resuspended in explant culture medium, and sorted using vYFP or GFP fluorescence on a Becton-Dickson FACS system (Rockville, Md.).