INTRODUCTION OF PLASMID DNA INTO ISOLATED-MITOCHONDRIA BY ELECTROPORATION

Mitochondrial disorders are a large group of phenotypically heterogene ous diseases. An understanding of their molecular basis would benefit greatly from the ability to manipulate the mitochondrial genome and/or to introduce functional exogenous DNA into mitochondria. As a first s tep toward this approach, we have used electroporation to introduce a 7.2-kilobase plasmid DNA into isolated functional mitochondria. Transf er of the DNA at field strengths between 8 and 20 kV/cm was investigat ed by Southern blot analysis. Maximal plasmid internalization was achi eved at a field strength of 14 kV/cm. The functional integrity of the mitochondria after electroporation was verified by enzymatic assays of specific mitochondrial marker enzymes and by measuring respiratory co ntrol. At field strengths above 12 kV/cm, an increasing mitochondrial destruction was observed. 12 kV/cm was found to be optimal for the mos t efficient plasmid internalization while still retaining the function al integrity of the mitochondria. At this field strength, about half o f the internalized plasmid was found in the inner membrane or mitochon drial matrix, as determined by immunoelectron microscopy and Southern blot analysis of electroporated mitochondria treated with digitonin. W e estimate that on average one plasmid molecule/mitochondrion reaches the matrix or inner membrane.