MiniVectors Used as a Template for Gene Repair

mv-CMV-TurboGFP: Gave 6, 5ug samples at 220 ng/µl in TE buffer, pH 8.0

Experiment courtesy of:
Ivette Sandoval, Ph.D.
Baylor College of Medicine

MiniVector encoding a fluorescent reporter protein was electroporated into the retina of neonatal mice. Expression of TurboGFP was evaluated by confocal microscope at 4 weeks post electroporation.

Methods

The 6, 5µg MiniVector samples were combined and ethanol precipitated. They were then resuspended in water at a concentration of 2.5 µg/µL. Electroporation of the MiniVector into the retina of neonatal mouse was performed shortly post birth. At 4 weeks post treatment, the mice were sacrificed and retinas processed as whole mounts. Confocal microscopy was used to evaluate the distribution of the TurboGFP in the retinal cells.

Results

MiniVector

Discussion

Expression of TurboGFP was observed in multiple cells of the neonatal retina. Cell morphology indicated a neuronal phenotype for many of the positive cells. Confocal z-stacks of the retina showed GFP expression in multiple layers of cells below the retinal surface.

A follow-up experiment was planned to compare the expression between MiniVectors and plasmids in the same experimental system. For this study, 20 µg TurboGFP expressing MiniVector and 20 µg TurboRFP expressing plasmid was provided. The two vectors are to be electroporated simultaneously into the same neonatal retina. Comparison of red and green expression will be performed at 4 weeks post electroporation.