The goal of this study was to determine whether the jellyfish
green fluorescent protein (GFP) could be used in transgenic
mice to label and purify cone photoreceptors from the living
retina. We created a transgene containing the 5′ regulatory
sequence of the human red pigment gene (pR6.5 lacZ clone; kindly
provided by J. Nathans & Y. Wang), fused to the GFP coding
sequence. This transgene was used to generate seven lines of
PCR-positive founders. Three of the lines had bright green
fluorescent cone photoreceptors. The GFP fills the entire cell.
Two mouse lines had only a few (∼10–100) fluorescent
cells per retina, and one line (R6.85933) had many thousands.
In the latter, double labeling of the cones with RITC-conjugated
peanut agglutinin reveals that in the ventral retina a small
proportion of the cones express GFP, while in the dorsal retina
the majority do. Cells dissociated from the retinae of line
R6.85933 continue to fluoresce and can be readily detected and
enriched with flow cytometry. The signal provides a log unit
of separation between the fluorescent cone soma and the remaining
retinal cells. Roughly 3% of the cells are this fluorescent,
and it is possible to purify up to 30,000 cells from one mouse.
RT-PCR analysis of the mRNA from these isolated cells detects
both the middle and short wavelength opsins with little if any
contamination from rhodopsin.