Biologically active human interferon alpha-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 alpha-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.

Human embryonic stem cell lines derived from discarded embryos.

Human pluripotent embryonic stem (ES) cells have important potential in regenerative medicine and as models for human preimplantation development; however, debate continues over whether embryos should be destroyed to produce human ES cells. We have derived four ES cell lines on mouse embryonic fibroblast cells in medium supplemented with basic fibroblast growth factor, human recombinant leukemia inhibitory factor, and fetal bovine serum. The source of these cell lines was poor-quality embryos that in the course of routine clinical practice would have been discarded.

Validating the hen as a bioreactor for the production of exogenous proteins in egg white.

Increased demand for the production of human biopharmaceuticals in transgenic organisms has led to an intensive effort to develop the hen as a bioreactor producing exogenous proteins in egg white via transgenesis. To date, however, robust methods for transgenic modification of the avian genome have been lacking. We have used a replication-defective retroviral vector derived from avian leukosis virus (ALV) to generate transgenic chickens expressing bacterial beta-lactamase secreted into serum and egg whites through several generations.

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, beta-lactamase, under the control of the ubiquitous cytomegalovirus (CMV) promoter. Biologically active beta-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.

Consistent production of transgenic chickens using replication-deficient retroviral vectors and high-throughput screening procedures.

We have developed a novel method of DNA extraction combined with a high-throughput method of gene detection allowing thousands of potentially transgenic chicks to be screened quickly and reliably. By using this method and a replication-deficient retroviral vector based on avian leukosis virus (ALV), we have demonstrated germline transmission of three different transgenes. Several generations of chickens carrying intact transgenes were produced, validating the use of the ALV retroviral vectors for large-scale production of transgenic flocks.