The lack of cone cell loss coupled with functional defects in GARP2-Tg cones led us to hypothesize that overexpression of GARP2 led to a physiological or signal transduction deficit in cones. However such a hypothesis would require expression of the GARP2 transgene in cones. Previous work has shown that a smaller 500-bp fragment of the mouse opsin promoter leads to rod expression and some leaky expression in cones.
28,29 Because previous studies did not verify whether the 4.4-kbp mouse opsin promoter used to generate the GARP2-Tg line could drive gene expression in cones,
30 we evaluated cone expression of the GARP2 transgene. P30
rds+/+ and GARP2-Tg/
rds+/+ retinal sections were colabeled with either S- or M-opsin (
Figs. 7A–
C, green) and GARP-4B1, which recognizes transgenic GARP2 as well as endogenous CNGB1/GARP2/GARP1 (
Figs. 7A,
7B, red) or c-Myc, which recognizes only transgenic GARP2 (
Fig. 7C, red). Single planes from a confocal image stack are shown. Although GARP-4B1 recognizes both transgenic and endogenous protein, it can be used to specifically assess transgene expression in cones because endogenous CNGB1/GARP2/GARP1 are not expressed in cones (
Figs. 7A,
7C,
7E, arrows and
Supplementary Fig. S3A show wild-type cones clearly not labeled with GARP-4B1 or c-Myc). In GARPT2-Tg/
rds+/+ retinas, most cone photoreceptors had no detectable GARP2 (
Figs. 7B,
7D,
7F, arrows). However, occasionally cone OSs were found in GARP2-Tg/
rds+/+ retinas that did label with the GARP-4B1 antibody (
Fig. 7A, arrowheads) suggesting there could be a low level of leaky GARP2 expression in cones in transgenic animals. Additional GARP2-Tg/
rds+/+ but not
rds+/+ cone OSs showing labeling with GARP-4B1 are pictured in
Supplementary Figure S3A and S3B (arrowheads). In
Supplementary Figures S3A, S3B, GARP-4B1 labeling is shown in red with cone OSs highlighted in green using the cone matrix sheath marker peanut agglutinin-PNA, and in each panel, the bottom row shows magnified insets of the cells highlighted by arrows in the top row. When we repeated this experiment using c-Myc antibody to specifically label transgenic GARP2, similar results were observed: some cone OSs did not express the transgene (
Fig. 7F, e.g., arrows) whereas others did (
Supplementary Fig. S3C, arrowheads). In many cases, the small size of murine OSs coupled with the high levels of GARP2 expression in adjacent rods made it difficult to determine whether cone cells had any detectable GARP-4B1 immunolabeling. These results suggest that while many cones in GARP2-Tg/
rds+/+ do not express any GARP2 transgenic protein; there are cones which exhibit ectopic expression. In addition, co-labeling with cone opsins and c-Myc (to specifically label GARP2-Tg) (
Fig. 7C) also confirmed that the GARP2 accumulation we observed in
Figure 2 was occurring in rods and not solely due to ectopic expression in cones.