Applications of Tol2 transposon-mediated gene transfer for stable integration and conditional expression of electroporated genes in chicken embryos

Because of the high accessibility to developing embryos, avian embryos (chicken and quail) have long been used as a good model animal to study embryogenesis in vertebrates, especially amniotes (reviewed in Wolpert, 2004). The techniques used for classical avian embryology included tissue transplantations, tissue ablations, and cell-labeling by vital dye. At the end of the last century, the in ovo electropora tion technique was developed by Nakamura and his colleagues, and this modern method opened a way to study the roles of developmental genes directly in living embryos (Funahashi et al., 1999) reviewed in (Nakamura et al., 2004; Yasuda et al., 2000; Yasugi and Nakamura, 2000). This powerful technique allows us to introduce genes (DNA, RNA, morpholino) into embryos in a tissue-specific way by targeting a restricted area of embryonic tissues. Thus, the electroporation technique using chickens has provided numerous novel insights into the understanding of early development in vertebrates, making the chicken a unique model animal. One of few shortfalls of the original technique has been that expression of electroporated genes does not persist for a long period of time probably because the introduced plasmids, which are not integrated into the genome, degrade or become diluted as embryonic cells undergo massive proliferation. Although a spontaneous genomic integration of electroporated genes could occur, this incidence must be extremely low. Since in most cases the electroporation is performed at embryonic day 1∼2 (E1∼E2), the short life of introduced genes hampers the analysis of the effects by introduced genes at late stages, i.e., from E5 onward, when a variety of organogenesis proceeds. At these late stages, the electroporation is difficult to perform because the embryo is much less accessible.