Electroporation

Takahiko Matsuda (Postdoctoral Fellow)
Claudio Punzo (Postdoctoral Fellow)

Electroporation can be carried out in vivo or in vitro, on chick, mouse, or rat retinas. This method has advantages over that of viral transduction and over trangenic animal production. Several (at least 5) plasmids can be simultaneously delivered, with regulated expression from each of the plasmids. A solution with a high concentration of plasmids is injected into the subretinal space and short pulses of high voltage are applied. The DNA is likely passively taken up via transient holes created in the membrane by the high voltage. Thus, only cells immediately adjacent to the injection site are transduced, and there appears to be no specificity in the uptake. For postnatal day 0-7 mouse and rat retinas, this means that progenitors that make rods, bipolars, amacrines and Muller glial cells are transduced, along with newly postmitotic cells that will achieve these fates. We have also been able to deliver plasmids in vivo to embryonic mouse retinas, with the most bright labeling of cells that become postmitotic shortly after deliver (e.g. cones, horizontal cells, amacrine cells). We have been unsuccessful transducing cells from the vitreal side, or in transducing adult cells from the scleral side. Electroporation of neonatal mouse and rat pups leads to expression for at least 50 days. Electroporation of rapidly dividing early chick retinal cells, e.g. at stage 10, leads to expression for only 2-3 days, likely due to dilution, and possibly degradation, of the plasmids. Plasmids encoding genes for gain-of-function studies have been successfully used. In addition, plasmids encoding RNA hairpins for RNAi have been successfully used. For studies of cis-acting regulatory regions, we have also been able to use plasmids with regulated promoters to begin to determine the sequences required for proper cell type specific expression (Figure 1).

Detailed protocols for electroporation by Takahiko Masuda and Claudio Punzo are available upon request. Maps of the vectors created by T. Matsuda for use in electroporation studies are also available, as are the plasmids themselves from Addgene (http://www.addgene.org/pgvec1?f=c&cmd=showcol&colid=116).

See our Publications page for pdf’s of the relevant manuscripts (Matsuda and Cepko, 2004, 2007, 2008 and Punzo and Cepko, 2008)