We report here the generation of transgenic chickens using a retroviral vector for the production of recombinant proteins. It was found that the transgene expression was suppressed when a Moloney murine leukemia virus-based retroviral vector was injected into chicken embryos at the blastodermal stage. When a concentrated viral solution was injected into the heart of developing embryos after 50 to 60 h of incubation, transgene expression was observed throughout the embryo, including the gonads. For practical production, a retroviral vector encoding an expression cassette of antiprion single-chain Fv fused with the Fc region of human immunoglobulin G1 (scFv-Fc) was injected into chicken embryos. The birds that hatched stably produced scFv-Fc in their serum and eggs at high levels (ϳ5.6 mg/ml). We obtained transgenic progeny from a transgenic chicken generated with this procedure. The transgene was stably integrated into the chromosomes of transgenic progeny. The transgenic progeny also expressed scFv-Fc in the serum and eggs.Transgenic bioreactors possess great potential for the production of recombinant pharmaceutical proteins (10,15,34). Transgenic animals have been generated for the production of recombinant proteins in the milk of livestock species such as goats, sheep, pigs, and cows. A high level of expression, more than 1 mg/ml of milk, has been reported with some proteins, and efforts toward a practical application for pharmaceutical use have continued (15). However, mammalian systems have several drawbacks in that they require a relatively large area for breeding and a long period for sexual maturation. As an alternative transgenic bioreactor, avian species such as chickens and quails have attracted a great deal of attention (11, 37). In particular, chickens have several advantages, including high protein productivity in eggs, ease of and small space requirements for breeding, similarity of the glycosylation pattern of proteins to that of humans (31), and absence of the prion problem.To date, many approaches have attempted to generate transgenic birds (38). Efforts have mainly involved either the injection of retroviral vectors into embryos at the blastodermal stage (1,7,8,24,26,32,35,36,42,43) or the microinjection of DNA into fertilized eggs at the single-cell stage (21, 39). Recently, lentiviral vectors were also used to generate transgenic chickens (3, 23). In most previous studies, a reporter gene such as lacZ and GPF has been used as the target, and expression was limited within cells. Harvey et al. reported that ␤-lactamase was produced in the serum and egg white of transgenic chickens generated with an avian leukosis virus-based retroviral vector (7,8). As a practical model for the production of a pharmaceutical protein, human alpha-2b interferon was produced using the same system (32). However, the expression levels in serum and eggs were not high enough compared with the mammalian transgenic bioreactor systems.Because of their availability, laid fertilized eggs at the blastodermal stage (stage X...

No abstract

The establishment of avian embryonic culture is important both for the analysis of the developmental process and the establishment of transgenic chickens that produce useful biological materials in eggs. However, the hatchability of cultured embryos has been approximately 50%. We identified that the low rate of hatchability of cultured embryos was caused by limited oxygen and calcium availability. In quail embryo culture using chicken eggshell as a culture vessel, viability in the middle stage of culture was improved and 30% of embryos were hatched by oxygen enrichment. Furthermore, hatchability increased to 80% by supplementation with calcium lactate in addition to oxygen aeration. In the present study, a fully artificial vessel for quail embryo culture was designed using a gas-permeable Teflon membrane. By the addition of fine eggshell powder and calcium lactate, quail embryos grew and developed normally, and 43% of embryos hatched. Although the hatchability was lower than that of cultures using a surrogate eggshell, we achieved in hatching an avian embryo using a fully artificial vessel.

An online system using HPLC was developed for the measurement of glucose, glutamine, and lactate in a culture broth. Using the system, the glucose and glutamine concentrations were controlled simultaneously by an adaptive-control algorithm within the ranges of 0.2 to 2.0 and 0.1 to 0.6 g/L, respectively. When the glucose concentration was controlled at the low level of 0.2 g/L, the intracellular lactate dehydrogenase activity decreased by one-half and the lactate concentration by one-third, whereas the uptake rates of serine and glycine were about twice as high, compared with the amounts when the glucose concentration was controlled at 1.0 g/L. On the other hand, ammonia production increased when the glucose concentration was kept low. To reduce the production of inhibitory metabolites such as ammonia and lactate and improve the antibody production rate in a hybridoma cell culture, the concentrations of glucose and glutamine were controlled at 0.2 and 0.1 g/L, respectively. With these low concentrations of glucose and glutamine, the cell concentration (4.1 x 10(6) cells/mL) and antibody production (172 mg/L) both increased about twofold compared with the amounts when the glucose was controlled at higher levels. From these results, simultaneous control of the glucose and glutamine concentrations was shown to be useful in the production of antibody by hybridoma cell cultivation.

No abstract

No abstract

A new type of aqueous two-phase system (ATPS) has been developed in which a temperature-sensitive polymer, poly-N-isopropylacrylamide [poly (NIPAM)] was used as a ligand carrier for the specific separation of animal cells. Monoclonal antibodies were modified with itaconic anhydride and copolymerized with N-isopropylacrylamide, and the ligand-conjugated carriers were added to the polyethylene glycol 8000-dextran T500 aqueous two-phase systems. The antibody-polymer conjugates were partitioned to the top phase in the absence or presence of 0.15 M NaCl. When ligand-conjugated carriers were used, more than 80% of the cells were specifically partitioned to the top phase in the presence of NaCl up to 0.1 M. The cells were partitioned almost completely to the bottom phase at 0.1 M NaCl or above, when no antibody-conjugate was added in the ATPS. As a model system, CD34-positive human acute myeloid leukemia cells (KG-1) were specifically separated from human T lymphoma cells (Jurkat) by applying anti-CD34 conjugated with poly-N-isopropylacrylamide in the aqueous two-phase system. By the temperature-induced precipitation of the polymer, about 90% of the antibody-polymer conjugates were recovered from the top phase, which gave approximately 75% cell separating efficiency in the next cycle of reuse.

No abstract