Generation of Transgenic Chickens Producing Human Erythropoietin.

The use of livestock animals as "bioreactors" to address the growing demand for large quantities and increasing numbers of biopharmaceuticals is of prime strategic relevance to agricultural improvement and medical advancement. We report here the production of transgenic chickens that produce human erythropoietin (hEPO) using replication-defective Moloney murine leukemia virus (MoMLV)-based vectors. Because it is well known that constitutive over-expression of some cytokine genes in the transgenic animals may cause serious physiological disturbances, the vectors were designed to express in the presence of tetracycline. In addition, we introduced WPRE (woodchuck hepatitis virus posttranscriptional regulatory element) sequence at 3' end of hEPO gene to boost the gene expression under the inducible condition. In a preliminary experiment in vitro, transformed primary chicken cells released hEPO into the medium as high as 30 μg/ml when the cells were cultured with doxycycline (1 μg/ml), a tetracycline derivative. Compared with the same cells not treated with doxycycline, fold induction in terms of hEPO expression was calculated to be more than eight. In a subsequent experiment, the recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). Out of 495 injected eggs, 25 chicks hatched after 21 days of incubation and 14 hatched chicks were found to express vector-encoded hG-GSF gene when fed with doxycycline. Quantitative analysis of the blood samples taken from some Go transgenic chickens resulted in as much as 7 μg /ml of hEPO in the blood. No physiological abnormality was observed from the transgenic chickens; therefore, this controllable gene expression system can probably expected to minimize possible detrimental side effects in the transgenic animals. The significance of this work stems from the fact that it is the first successful report on the production of transgenic chickens expressing the hEPO gene. This approach can be employed to create a useful transgenic model system for further studies on the chicken embryo development and the efficient production of transgenic chickens as bioreactors.[This work was supported by: The BioGreen 21 Program (Grant No. 20050501-034-844) of the Rural Development Administration, Republic of Korea; The SRC/ERC program of MOST/KOSEF (grant No. R11-2002-100-01005-0); The 2006-2011 Technology Development Program for Agriculture and Forestry (TDPAF), Ministry of Agriculture and Forestry, Republic of Korea.]