Figure 6.

Lens-specific and ubiquitous expression of RYBP/EGFP fusion protein. (A) Scheme illustrating the strategy used to generate a Cre recombinase-mediated conditional ectopic Rybp allele by targeting the ROSA26 locus (for details, see Methods). The position of the hybridization probe (dot) and restriction enzyme EcoRV (arrows) used for detecting the correct homologous integrants by Southern blotting is shown. The 5' probe used detects an 11 kb wt band and a 3.8 kb targeted band, due to the presence of an extra EcoRV site in the targeted allele. The exons shown as open boxes. Cre: Cre-recombinase, EGFP; enhance green fluorescent protein, Neo; neomycin phosphotransferase. (B) Western blotting analysis showing the expression of the RYBP/EGFP fusion in lysates of cells that have been exposed to Cre recombinase. Lane 1, lysates from ROSA26 knock-in ES cells prior to Cre exposure (-Cre); lane 2, lysates from Rosa26 knock-in ES cells after Cre exposure (+Cre); lane 3, lysates from ES cells transfected with an RybpFlag construct, as a positive control. The blot was probed with the anti-Rybp antibody. (C) Ectopically expressed Rybp can associate with Ring1A. Lanes 1–2: Cells lysates of transfected ES cells blotted with anti-Flag antibody showing that both Ring1aFlag (Lane 1) and Maf1Flag (Lane 2) strongly expressed in the transfected cells; Lane 1, Cells transfected with Ring1AFlag construct., Lane 2, Cells transfected with Maf1Flag construct. Lane 3–4: Cells transfected with either Ring1AFlag (Lane 3) or with MAF1Flag (Lane 4), then immunoprecipitated with Rybp and blotted with anti-Flag antibody. Lane 3 shows the 57 kdDa Ring1AFlag fusion protein as the result of the association with the RYBP/EGFP fusion protein. Maf1 cannot bind Rybp (Lane 4). (D) P1 eyes harvested from double transgenic (ROSA26-RYBP/EGFP; αA-crystallin/Cre mice) and wildtype (WT) animals shown in dark field and fluorescence. (E) Lens specific over-expression of the RYBP/EGFP fusion protein in newborn (P2) lenses. EGFP immunohistochemistry shows the overexpression of the transgene in the ROSA26-RYBP/EGFP; αA-crystallin/Cre lens (TG). The wild-type lens shows only background staining (WT). (F) Bright-field and dark-field pictures of E11.5 littermates. All embryos that genotyped double transgenic for ROSA26-RYBP/EGFP;β-Actin/Cre also exhibited detectable EGFP fluorescence (TG) when compared to wildtype (WT) littermates. Magnification: D (×40); E (×10)

Pirity et al. BMC Developmental Biology 2007 7:39   doi:10.1186/1471-213X-7-39
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