Gordon Speksnijder

Expression of exogenous protein in the egg white of transgenic chickens.

Using a replication-deficient retroviral vector based on the avian leukosis virus (ALV), we inserted into the chicken genome a transgene encoding a secreted protein, β-lactamase, under the control of the ubiquitous cytomegalovirus (CMV) promoter. Biologically active β-lactamase was secreted into the serum and egg white of four generations of transgenic chickens. The expression levels were similar in successive generations, and expression levels in the magnum of the oviduct were constant over at least 16 months in transgenic hens, indicating that the transgene was stable and not subject to silencing. These results support the potential of the hen as a bioreactor for the production of commercially valuable, biologically active proteins in egg white.

Biologically Active Human Interferon α-2b Produced in the Egg White of Transgenic Hens

We have previously described the expression of a bacterial protein in the egg white of transgenic chickens using a replication-deficient retroviral vector. Here we report the expression of a glycosylated human protein, interferon α-2b (hIFN), in the egg white of transgenic hens. The hIFN secreted into the egg white was biologically active as determined by a viral inhibition assay. Purification and carbohydrate analysis of the hIFN expressed in egg white revealed that two of the six major glycosylated hIFN species match the naturally occurring human hIFN glycovariants. These results support the potential of the hen as a bioreactor for the production of commercially valuable, biologically active, and glycosylated proteins in egg white.

Progress toward the culture and transformation of chicken blastodermal cells.

Chicken blastodermal cells can be cultured for short periods of time and retain the ability to contribute to somatic and germline tissues when injected into γ‐irradiated stage X embryos. Such a method has yet to yield a germline transgenic bird, in part due to the low rate of transgene integration into the avian genome. In addition, the short culture period precludes the identification and expansion of those cells that carry an integrated transgene. In this study, two methods were developed that produced blastodermal cells isolated from stage X Barred Plymouth Rock embryos bearing an integrated transgene. Addition of chick embryo extract to the culture medium enabled expansion of single colonies for multiple passages. Southern blot analysis indicated that the transgenes had integrated as a single copy in most of the clones. Cells from passaged, transgenic embryo cells were injected into irradiated stage X White Leghorn embryos, producing hatched chicks that bore the donor cells in their somatic tissues. Transgene sequences were detected in sperm DNA; however, breeding of chimeras did not result in germline transmission of the transgene, indicating that the contribution of the transgenic cells to the germline was either nonexistent or very low.