Transgenic expression of the jellyfish green fluorescent protein in the cone photoreceptors of the mouse.
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.
Fei
Y
Y
Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, CT 06520, USA.
Hughes
T E
TE
D000818:Animals; D019677:Artificial Gene Fusion; D005136:Eye Proteins / Q000235:genetics; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D017949:Retinal Cone Photoreceptor Cells / Q000502:physiology*; D017299:Rod Opsins; D019076:Transgenes
D005136:Eye Proteins; D008164:Luminescent Proteins; D017299:Rod Opsins; C095855:long-wavelength opsin; D049452:Green Fluorescent Proteins