Using electroporation to transiently express plasmid DNA in Ciona embryo. The Ciona system allows rapid delivery and expression of reporter gene constructs in the developing embryo. It is also convenient to use tissue-specific enhancers for targeted misexpression in specific embryonic lineages. (A) Eggs and sperm are isolated from the adult for in vitro fertilization. The fertilized eggs are dechorionated, mixed with plasmid DNA and electroporated at the 1-cell stage. The electroporated embryos are then allowed to develop to appropriate developmental stages for reporter gene analysis (X-gal staining) or fixed for in situ hybridization. (B) Schematic of serial deletion analysis. The last construct depicts the minimal enhancer element (solid line) activating reporter gene expression with a basal promoter. It often requires testing of 20-40 deletion constructs to identify a minimal enhancer. (C) Schematic of site-directed mutagenesis of a minimal regulatory element. Shapes represent different putative transcription factor binding sites, filled for wild-type and unfilled for mutated. In this case, the binding sites represented by diamonds were required for reporter expression. Thorough characterization of a minimal enhancer through this method would also require testing of at least 20-40 constructs. Because one can test 10-16 constructs in a single round of electroporations, a single researcher can test over 100 constructs/week. Thus, in-depth serial deletion (B) and subsequent site-directed mutagenesis (C) can be conducted over a few weeks rather than many months.
