Figure 5

Cryab promoter without GPS (ΔGPS) is inactive in transgenic mice. (A) Schematic of the + GPS cryab promoter-tGFP transgene (GPS, blue rectangle). Primer locations for genotyping are shown. (B) Genotyping of transgenic mice, F forward primer, R reverse primer. Note the lower mobility of the PCR product (270 bp, left panel) generated from ΔGPS mice in comparison with the (+)GPS mice (280 bp). The right panel (with 2F and 2R, internal for tGFP) shows no change in amplicon size (290 bp). WT wild-type non-transgenic DNA, Blk blank. (C) Expression of tGFP (anti-tGFP immunofluorescence, confocal images) in three transgenic mouse lines (numbers 1–3) in + GPS and ΔGPS mice. Middle z sections of three tissues (eye, heart, and liver) are shown from + GPS (top) and ΔGPS mice (bottom). In + GPS transgenic tissues, tGFP immunofluorescence is obvious in both the developing heart (ve ventricle) and the eye (ocular lens (le), corneal epithelium (ce), and ganglion cells (gc)). In addition, tGFP expression is seen in the surrounding choroid and mesenchymal cells between the lens and the developing retina. The specific expression of tGFP in hepatic stellate cells (sc) is striking (+GPS, liver). Note the absence of expression (immunofluorescence) in the ΔGPS lines in all tissues (bottom). (D) Immunoblots of 11 tissue extracts of F1, (+)GPS and ΔGPS (transgenic line 2), and WT. Gapdh green bands (internal control). Le lens, Re retina, Eg eye globe without the lens and retina, Li liver, He heart, Lu lung, Sp spleen, Ki kidney, Si small intestine, Mu muscle, Br brain, C control ARPE-19 cell extract. (E) Immunoblots of tissue extracts as in (D) from F2, line 2 transgenic animals. Note that the WT and ΔGPS tissues do not show any reactivity for tGFP.