RESUMO
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 gamma-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.
Assuntos
Blastoderma/citologia , Blastoderma/efeitos da radiação , Técnicas de Cultura de Células/tendências , Animais , Blastômeros/fisiologia , Blastômeros/efeitos da radiação , Embrião de Galinha , Técnicas de Cocultura , Resistência a Medicamentos/genética , Eletroporação , Fibroblastos/metabolismo , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Células Híbridas , Camundongos , Plasmídeos/metabolismo , Puromicina/efeitos adversos , Extratos de Tecidos/farmacologia , Transfecção , TransgenesRESUMO
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. 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.
