The distribution, concentration, and toxicity of enhanced green fluorescent protein in retinal cells after genomic or somatic (virus-mediated) gene transfer.
OBJECTIVE
The concentration of enhanced green fluorescent protein (EGFP) in individual photoreceptor cells of live mouse retina was quantified and correlated with physiological measurements of cell function.


METHODS
EGFP protein levels in the retinas of mice injected subretinally by either one of two serotypes of adeno-associated virus (AAV; AAV2/5.CMV.EGFP; AAV2/2.CMV.EGFP) were quantified with a photon-counting confocal laser scanning microscope and compared with those of transgenic mice whose retinas expressed EGFP under the beta-actin (pbetaAct) or human L/M-cone opsin (pLMCOps) promoter. Single-cell suction pipette recordings of single rods and whole-field electroretinograms (ERGs) were performed to assess retinal cell function.


RESULTS
The highest levels of EGFP (680 microM) were in the retinal pigment epithelium (RPE) cells of the AAV-transduced eyes. Living photoreceptors of pbetaAct.EGFP mice contained 270 microM EGFP, while their bipolars had 440 microM. The cones of pLMCOps.EGFP mice expressed 60 microM protein. The amplitudes of the major components of ERGs were within the normal range for all transgenic animals examined, and single cell recordings from living pbetaAct.EGFP rods were indistinguishable from those of controls.


CONCLUSIONS
EGFP levels in individual cells of live mouse retinas can be quantified, so that the efficacy of gene transfer methods can be quantified. Concentrations of several hundred microM are not deleterious to normal function of photoreceptors and bipolar cells. This approach can also be used to quantify levels of biologically active EGFP fusion proteins.
Rex
Tonia S
TS
F. M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania, Philadelphia, PA 19104, USA. trex@mail.med.upenn.edu
Peet
John A
JA
Surace
Enrico M
EM
Calvert
Peter D
PD
Nikonov
Sergei S
SS
Lyubarsky
Arkady L
AL
Bendo
Elisabeth
E
Hughes
Thomas
T
Pugh
E N
EN
Bennett
Jean
J
D000818:Animals; D000229:Dependovirus / Q000235:genetics; D004594:Electrophysiology; D004596:Electroretinography; D004622:Embryo, Mammalian / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000008:administration & dosage / Q000493:pharmacokinetics* / Q000506:poisoning*; D007267:Injections; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D009994:Osmolar Concentration; D010775:Photic Stimulation; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism; D012160:Retina / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism* / Q000502:physiology; D051245:Retinal Bipolar Cells / Q000378:metabolism; D014018:Tissue Distribution
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins