Purchase this article with an account.
F. A. Concepcion, S. S. Patel, M. A. Agosto, J. H. Wilson, T. G. Wensel, D. K. Mojumder; Development and Testing of a Low-Cost PC Controlled in vivo Electroporator. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3130.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Gene therapy involves the introduction of multiple factors into the targeted cells of interest. These factors include double strand DNA break- (DSB) inducers (such as zinc finger nucleases) and correcting donor DNA (for gene repair through homologous recombination). In vivo electroporation (Matsuda & Cepko 2004 PNAS 101:16) has been demonstrated as an effective method to introduce plasmids in rodent eyes. Here, we report the development and testing of a low-cost yet versatile, personal computer controlled electroporator which is capable of transfecting DNA into various tissues of the mouse eye.
The hardware consists of a circuit that regulates a constant voltage source controlled by a software with an easy and flexible graphical user interface. Plasmids encoding either eGFP or mCherry fluorescent proteins [driven by cytomegalovirus (CMV), murine opsins (MOPS), or metabotropic glutamate receptor 6 (mGluR6) promoters] were purified by using Qiagen maxipreps. Subretinal injections of different plasmids were performed in either neonatal pups (post natal day 0, P0) or mice after weaning. The electroporator settings were based largely on the parameters reported by Matsuda & Cepko (2004). Retinal and at times corneal whole mounts were performed to examine fluorescent protein expression.
With this low cost yet versatile electroporator, we were able to induce expression of either eGFP or mCherry, or both, in various murine eye tissues. In mice transfected after weaning, we detected CMV driven eGFP expression in the cornea (epithelial cells and keratocytes) and - albeit low levels - in the retina (bipolar, Müller, horizontal, and ganglion cells). From mice transfected at P0, we observed at a later time point: (1) expression of eGFP or mCherry, or both, in photoreceptors when driven by either the CMV or MOPS promoter, and (2) expression of eGFP in bipolar cells when driven by the mGluR6 promoter. When injections involved multiple plasmids, we observed cotransfection in several photoreceptors cells.
With this low-cost, PC-based design, in vivo electroporation technology can be made easily accessible to most research laboratories at a fraction of the cost of commercially available electroporators.
This PDF is available to Subscribers Only