Transgenic Chicken as Bioreactor for Producing Pharmaceutical Proteins

Abstract

The demand for pharmaceutical proteins is rapidly increasing in Indian bio-industry. These proteins are generally produced by genetic engineering of bacteria or mammalian cells and their large-scale culture of cells fermmentors or bioreactors. The production of such proteins is not only costly but also has low bio-activity. Consequently, transgenic animals are being viewed as a good alternative to bacterial or mammalian cell culture-based protein production. Among various animals, chicken is emerging as an excellent animal bioreactor for producing human proteins of pharmaceutical importance. They not only have short generation interval, low cost and high fecundity but also offer a unique advantage of ease in purifying the recombinant protein from eegg, which is biochemically less complex and contain -65% of albumen alone. Here, we describe the generation of transgenic chickens expressing human parathormone (hPTH), human urokinase- type plasminogen activator (huPA) or human epidermal growth factor (hEGF) for use as human drugs. The chickens were produced using a robust replication-defective Moloney murine leukemia virus-based retrovirus vector encapsidated with vesicular stomatitis virus G glycoprotein. Recombinant hPTH, huPA or hEGF genes were constructed to express the respected genes under the control of ubiquitous Cytomegalovirus (CMV) and Rous sarcoma virus (RSV) promoter. The recombinant retrovirus was injected into the subgerminal cavity of freshly laid eggs at the blastodermal stage by windowed egg method. After 21 days of incubation, hatched chicks expressed the vector-encoded hPTH, huPA or hEGF genes in diverse organs, as revealed by PCR and RT-PCR analysis. Four days after hatching, six hPTH expressing chicks died and 14 chicks showed phenotypic deformities. At 18 weeks of age, the survived chicks released high level of hPTH, huPA or hEGF proteins in their blood and transmitted the genes to Gl embryos. However, level of protein expression varied with the integrated gene and the gene copy number. These data demonstrate that transgenic chickens, expressing a human protein under the control ofa ubiquitous promoter, could not only be an efficient bioreactor for the production ofpharmaceutical drugs.