Live Detection of the Firefly Luciferase Gene Expression by Bioluminescence in Incubating Chicken Embryos

Abstract

By using in ovo electroporation, the firefly luciferase gene was transfected to living chicken embryos at 2 days of incubation.At 48hrs after transfection, expression of the firefly luciferase gene was monitored in ovo by using a single photon imaging counter.Strong bioluminescence detected from the incubating embryo suggests that in ovo electroporation would provide a useful and efficient nonviral means of foreign gene transfection to somatic cells of living chicken embryos.

In ovo electroporation of avian somites

“…The first successful electroporation of chick tissue was performed on retinal explants in vitro (Pu and Young, 1990). Muramatsu et al (1996Muramatsu et al ( , 1997 succeeded in electroporating chick embryos in ovo, and Momose et al (1999) optimized the technique to specifically target tissues of interest in ovo by the use of microelectrodes (reviewed in Atkins et al, 2000). Since then, many functional studies based on electroporation-mediated gene transfer in chick have been published.…”

Electroporation and EGFP labeling of gastrulating quail embryos

“…Tissue movements and deformations, while not as well studied, are equally important in understanding formation of the bilateral body plan of warm-blooded embryos (Fraser and Harland 2000;Keller et al, 2003;Czirok et al, 2004;Zamir et al, 2005). Embryologists have invented a number of techniques for selectively labeling live cells and/or tissues in order to study their movements (Graeper, 1929;Le Douarin, 1973;Muramatsu et al, 1996; for review: Stern and Fraser, 2001). The advent of genetically engineered fluorescent protein vectors is a powerful tool, particularly when coupled to modern microscopes and image detectors.…”

“…Techniques for electroporating bird embryos in ovo are well established (Muramatsu et al, 1996;Krull, 2004). The approach we devised requires removal of the embryo from the egg.…”

Electroporation: an arsenal of application

“…Firstly, this approach offers ways of genetic manipulation in animals that lack the methods for targeted transgenic studies (Swartz et al 2001). One of the most successful models was built with chicken embryos, which was also called In Ovo Electroporation and the first reported studies of in ovo electroporation used the firefly luciferase gene as the live reporter (Muramatsu et al 1996). Moreover, this method could provide a quick way of gaining or loss of function.…”

“…The hydrophilic pore was induced by electric pulses on human erythrocyte (Kinosita et al 1977a, b, c), and successful plasmid delivery to mammalian cells was achieved at 1982 Wong et al 1982). After that, electroporation models were built on both monocot and dicot plant cells (Fromm et al 1985(Fromm et al , 1986Ou-Lee et al 1986), bacteria cells (Schivarova et al 1983), yeasts (Hashimoto et al 1985;Karube et al 1985) and eventually embryos (Muramatsu et al 1996;Akamatsu et al 1999). The advantages of this method include precise transfection, higher efficiency when a smaller volume of materials was used, the fast detection of reporter genes and the less toxicity to tissues (Momose et al 1999).…”