| 1. |
Yoshiko Takahashi, Tadayoshi Watanabe, Shinichi Nakagawa, Koichi Kawakami, Yuki Sato, Chapter 14 Transposon-Mediated Stable Integration and Tetracycline-Inducible Expression of Electroporated Transgenes in Chicken Embryos, 10.1016/S0091-679X(08)00214-8, 87, 271-280, 2008.05, The Tol2-mediated transposition approaches are novel techniques for the molecular manipulation of chicken embryos by which an exogenous gene can be integrated into the host genome. The integrated gene(s) can be stably expressed when driven by a ubiquitous promoter. This method can also be used for conditional expression when combined with the Tet-on system to achieve temporal control. The Tol2-transposition method allows for expression at stages even later than E5, which otherwise could not be analyzed with the conventional electroporation technique. These developmental stages are critical for organogenesis, where numerous tissues interact to generate the complex structures of functional organs. Thus, the techniques presented here are extremely useful for understanding how cells contribute to organogenesis at the molecular level.. |
| 2. |
Yuki Sato, Yoshiko Takahashi, Applications of Tol2 transposon-mediated gene transfer for stable integration and conditional expression of electroporated genes in chicken embryos, Springer Japan, 10.1007/978-4-431-09427-2_3, 17-24, 2009, 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.. |
