336 QUANTITATIVE ANALYSIS OF TETRACYCLINE-INDUCIBLE EXPRESSION OF THE GREEN FLUORESCENT PROTEIN GENE IN TRANSGENIC CHICKENS

Abstract

The use of transgenic farm animals as bioreactors to address the growing demand for biopharmaceuticals, both in terms of increased quantity and greater number, represents a key development in the advancement of medical science. However, the potential for detrimental side effects as a result of uncontrolled constitutive expression of foreign genes in transgenic animals is a well-recognised limitation of such systems. Previously, using a tetracycline-inducible expression system, we demonstrated the induction of expression of a transgene encoding green fluorescent protein (GFP) in transgenic chickens by feeding with doxycycline, a tetracycline derivative; expression of GFP reverted to pre-induction levels when the inducer was removed from the diet (Kwon et al. 2011 Biochem. Biophys. Res. Commun. 410, 890–894). As a proof of principle study, however, quantitative assessment of expression was not possible, as only 1 G0 and 1 G1 transgenic chicken was obtained. In the current study, with 7 G2 transgenic chickens obtained from 1 G1 hen, we confirmed stable genomic integration of a single copy number of the transgene by Southern blot analysis. As we have observed in G1 transgenic chicken previously, all of the G2 transgenic chickens emitted a green fluorescence upon doxycycline feeding (50 mg kg^–1 of formula feed). Fluorescence became detectable 4 days after starting doxycycline feeding, and maximum GFP expression was detected after 2 weeks. Removal of doxycycline from the diet after 14 days of induction feeding resulted in the return of external fluorescence to pre-induction levels after 39 days. Quantitative analysis of gene induction was done using protein and mRNA extracted from primary cultured cells derived from 6-day transgenic chicken embryos. The eggs were obtained by mating a nontransgenic wild-type hen with 1 of G2 transgenic roosters. Protein levels of GFP were analysed by immunoblot and quantified using a densitometer. In the absence of doxycycline, the amount of GFP per 1 µg of total protein was 0.2 ng. However, when the cells were treated with doxycycline for 6 days, the amount of GFP increased to 3.1 ng per 1 µg of total protein, which was 16-fold higher than that of the cells pre-treated with doxycycline. Switching to doxycycline-free medium after doxycycline induction resulted in significant abrogation of GFP expression in 6 days; the amount of GFP reduced from 3.1 to 0.5 ng, a 6.2-fold reduction. Transcription of the GFP gene was also assessed by Northern blot. The amount of GFP mRNA measured by band density increased as much as 20-fold (3.9/0.2) with 6 days of doxycycline induction and declined to 1/8 (3.9/0.5) when doxycycline was removed from the cell culture media for 6 days. The use of an inducible expression system that can be regulated by dietary supplementation could help mitigate the physiological disruption that can occur in transgenic animals as a result of uncontrolled constitutive expression of a transgene.