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In this study, we report successful expression of recombinant human erythropoietin (hEPO) in the egg white of transgenic hens using a feline immunodeficiency virus (FIV)- based lentiviral vector as an exogenous gene deliverer. hEPO is a glycoprotein hormone that controls erythropoiesis, or red blood cell production. FIV vectors permit high levels of transgene expression in quails and chickens. We constructed a FIV vector containing a hEPO cDNA sequence driven by an oviduct-specific ovalbumin promoter, and microinjected into the subgerminal cavity of stage X chick embryos to generate transgenic chicken. Out of 208 injected eggs, 10 chicks were hatched after 21 days of incubation, and one of the G0 hatched chicken expressed the vector-encoded hEPO gene in sperm. Successful germline transmission of the transgene was also confirmed in G1 transgenic chicks produced from crossing G0 transgenic roosters with non-transgenic hens. One rooster was mated to wild-type hens to produce 518 G1 progeny. PCR analysis of blood samples from these progeny revealed that there were four G1 transgenic offspring, corresponding to a 0.77% germline transmission rate. Subsequently, Southern blot analysis of the genomic DNA from four G1 transgenic chickens was carried out to verify the stable genomic integration and copy number of the transgene in the genome. Quantitative analyses of the blood and egg white samples taken from G1 transgenic chickens resulted in 4,810～6,600 IU/mL (40.1～55.0 ㎍/mL) of hEPO in egg white, whereas 18～25 mIU/mL (0.15～0.2 ng/mL) in serum. The biological activity of the recombinant hEPO in egg white was comparable to its commercially available counterpart. We conclude that successful expression of recombinant hEPO in the egg white of transgenic chickens implies an important step towards efficient production of human cytokine from the transgenic animal bioreactor.