Schematic representation of I-SceI vector microinjection. Microinjection for meganuclease mediated transgenesis should be performed as shown. Embryos should be oriented as indicated. The injection volume must not exceed 10% of the cell volume. (a) An I-SceI vector. The insertion cassette contains an expression cassette that includes the gene of interest and a reporter cassette. Two insulator sequences and two inverted I-SceI recognition sites flank the entire insertion cassette. (b) Injection is performed directly into the cytoplasm of the cell. Upon co-injection of DNA with I-SceI, this procedure will significantly enhance transient transgene expression and frequency of transgenesis. Injection without I-SceI will result in highly mosaic transient transgene expression and low transgenesis frequency, even if injected into the cytoplasm. (c) Injection into the yolk of a one-cell-stage zebrafish abolishes the enhancing effect of I-SceI. Transgene expression and transgenesis frequency will be similar to those with conventional microinjection. Therefore, injection into the yolk only should be avoided. GOI, gene of interest; INS, insulator; P, promoter; pA, polyadenylation signal; REP, reporter gene. Modified from Grabher and coworkers .