In Vivo Electroporation of DNA into the Wing Epidermis of the Butterfly,Bicyclus anynana

Abstract

The direct transfer of genes into differentiated insect tissues is a useful method of determining gene function because it circumvents the need to perform germ line transformations and of having information on tissue-specific gene promoters. Here in vivo gene delivery is achieved through electroporation of a reporter gene into the pupal forewing of the butterfly Bicyclus anynana (Butler) (Lepidoptera: Nymphalidae). Plasmids containing the coding sequence for enhanced green fluorescent protein (EGFP), driven by the Drosophila heat-shock promoter hsp70, were successfully expressed in epidermal cells after plasmid injection followed by electroporation and heat shock. EGFP expression was restricted to the vicinity of the injection and electroporation site, but the number of transformed cells varied from a few to over 5000 cells. Electroporation parameters were optimized in order to maximize the number of transformed cells while minimizing the extent of damage to the adult wing. While certain electrical parameters were well tolerated by the wing tissue, the physical damage caused by the insertion of the tungsten electrodes led to frequent disruptions of the adult wing pattern around the puncture sites. While this technique can be useful to test the correct expression of marker genes (such as EGFP) in newly build plasmids immediately following their injection, its potential use in testing the function of candidate genes in wing pattern formation is limited.