Temporal Control of Gene Expression by Combining Electroporation and the Tetracycline Inducible Systems in Vertebrate Embryos
The electroporation technique has revolutionized vertebrate embryology. It has greatly contributed to our understanding of how genes and proteins can interact and regulate various aspects of vertebrate development in the last decade. This technique provides an efficient way to transfect embryonic cells in vivo with exogenous DNA by cre ating transient holes in the plasma membrane with short, squared electric pulses of low voltage (Itasaki et al., 1999; Momose et al., 1999; Muramatsu et al., 1997; Nakamura et al., 2004; Ogura, 2002). It has been particularly well-developed in the chick model since the large size of the embryo and its easy accessibility enables to target specific tissues with great precision. With the electroporation, it is possible to precisely choose which type of cells to transfect by performing a local injection of DNA close to the cells of interest, followed by the application of a small current through the targeted area. To date, all three germ layers — endoderm, mesoderm and ectoderm — as well as an increasing number of differentiated structures have been efficiently transfected (Dubrulle et al., 2001; Grapin-Botton et al., 2001; Itasaki et al., 1999; Luo and Redies, 2005; Scaal et al., 2004) and the continuous improvement in electrode design makes it even possible to aim at sub-populations of cells within a given tissue. In addition to this spatial precision, the technique also allows great temporal precision; any stage of development, ranging from pre-gastrulation stage to adulthood can be reached as long as the cells or structures are accessible for local DNA injection and electrode placement (Bigey et al., 2002; Iimura and Pourquie, 2006).
A drawback of this technique is that such electroporations lead to a sustained over expression of the transgene until the DNA gets diluted out or degraded, which can take 2 days or even more, depending on the rate of cell proliferation of the targeted tissue. This prolonged overexpression may have cumulative effects that can obliterate the primary role of the studied molecule in a given process at the time the phenotype is being assessed. This is especially worrisome, since, during early development, signaling molecules and growth factors are usually transiently expressed and act during short time windows. In addition, signaling molecules or transcription factors are often used repeatedly at multiple steps during morphogenetic events. Artificially prolonged exposure as a result of electroporation may blur their role during a given step or lead to aberrant cellular behaviors. There is therefore definitely a need to control the timing of expression of the transgene in the embryo. Several strategies have been developed over the past decades to control gene expression in time and space. Most of these strategies are based on genetic tools developed in fly and mouse, like the UAS-Gal4 system or the Cre-Lox induced recombination system. However, because manipulating the avian genome is yet unrealistic, those powerful strategies cannot be applied. Alternative inducible systems, based on prokaryotes operons, are available to control gene expres sion in time without genetic manipulation, and the most versatile one is probably the tetracycline inducible system (Tet-Off and Tet-On) developed by Gossen et al. more than 15 years ago (Gossen and Bujard, 1992). This chapter describes how the tetra cycline inducible systems can be applied to avian embryos using the electroporation technique and what kind of applications one can expect from them.
KeywordsNeural Tube Chicken Embryo Primitive Streak tetR Protein Presomitic Mesoderm
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