Hubdar Ali Kaleri

Egg-laying hens are important candidate bioreactors for pharmaceutical protein production because of the amenability of their eggs for protein expression. In this study, we constructed an oviduct-specific vector containing tissue plasminogen activator (tPA) protein and green fluorescent protein (pL-2.8OVtPAGFP) and assessed its expression in vitro and in vivo. Oviduct epithelial and 3T3 cells were cultured and transfected with pL-2.8OVtPAGFP and pEGP-N1 (control vector), respectively. The pL-2.8OVtPAGFP vector was administered to laying hens via a wing vein and their eggs and tissues were examined for tPA expression. The oviduct-specific vector pL-2.8OVtPAGFP was expressed only in oviduct epithelial cells whereas pEGP-N1 was detected in oviduct epithelial and 3T3 cells. Western blotting detected a 89 kDa band corresponding to tPA in egg white and oviduct epithelial cells, thus confirming expression of the protein. The amount of tPAGFP in eggs ranged 9 to 41 ng/mL on the third day after vector injection. The tPA expressed in egg white and oviduct epithelial cells showed fibrinolytic activity, indicating that the protein was expressed in active form. GFP was observed only in oviducts, with no detection in heart, muscle, liver and intestine. This is the first study to report the expression of tPA in egg white and oviduct epithelial cells using an oviduct-specific vector.

We successfully replaced the ovalbumin gene of a magnum region in chickens with a human plasminogen activator. We constructed pL-eGFP, pL-tPAGFP and pL-2.8OVtPAGFP vectors and cultured 293FT chicken embryo fibroblasts, chicken primordial germ cells, Hela C127 cells, and oviduct epithelial cells. All vectors were expressed in the transfected cells, except pL-2.8OVtPAGFP vector, which was only expressed in oviduct epithelial cells. A lentivirus with pL-2.8OVtPAGFP was injected in fertilized eggs; 11 chicks hatched in the G₀ generation, four of them carried the tPAGFP. Two cockerels from the G₀ generation were crossed with four wild-type hens. Three chicks in G₁ carried the tPAGFP. We concluded that by using an oviduct-specific vector for transfection, human recombinant plasminogen activator protein can be expressed in the oviducts of laying hens. This character is inherited and can be reproduced with a need for repeated transfection.