RESUMO
Primordial germ cells (PGCs) are the precursors of sperm and eggs. In most animals, segregation of the germ line from the somatic lineages is one of the earliest events in development; in avian embryos, PGCs are first identified in an extra-embryonic region, the germinal crescent, after approximately 18 h of incubation. After 50-55 h of development, PGCs migrate to the gonad and subsequently produce functional sperm and oocytes. So far, cultures of PGCs that remain restricted to the germ line have not been reported in any species. Here we show that chicken PGCs can be isolated, cultured and genetically modified while maintaining their commitment to the germ line. Furthermore, we show that chicken PGCs can be induced in vitro to differentiate into embryonic germ cells that contribute to somatic tissues. Retention of the commitment of PGCs to the germ line after extended periods in culture and after genetic modification combined with their capacity to acquire somatic competence in vitro provides a new model for developmental biology. The utility of the model is enhanced by the accessibility of the avian embryo, which facilitates access to the earliest stages of development and supplies a facile route for the reintroduction of PGCs into the embryonic vasculature. In addition, these attributes create new opportunities to manipulate the genome of chickens for agricultural and pharmaceutical applications.
Assuntos
Linhagem da Célula , Galinhas/genética , Células Germinativas/citologia , Células Germinativas/metabolismo , Mutação em Linhagem Germinativa/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Embrião de Galinha , Feminino , Citometria de Fluxo , Engenharia Genética/métodos , Genoma/genética , Células Germinativas/transplante , Cariotipagem , Masculino , Óvulo/citologia , Óvulo/metabolismo , Espermatozoides/citologia , Espermatozoides/metabolismo , Transplante de Células-TroncoRESUMO
Male and female embryonic stem (ES) cell lines were derived from the area pellucidae of Stage X (EG&K) chicken embryos. These ES cell lines were grown in culture for extended periods of time and the majority of the cells retained a diploid karyotype. When reintroduced into Stage VI-X (EG&K) recipient embryos, the cES cells were able to contribute to all somatic tissues. By combining irradiation of the recipient embryo with exposure of the cES cells to the embryonic environment in diapause, a high frequency and extent of chimerism was obtained. High-grade chimeras, indistinguishable from the donor phenotype by feather pigmentation, were produced. A transgene encoding GFP was incorporated into the genome of cES cells under control of the ubiquitous promoter CX and GFP was widely expressed in somatic tissues. Although cES cells made extensive contributions to the somatic tissues, contribution to the germline was not observed.
Assuntos
Embrião de Galinha/citologia , Quimera , Células-Tronco Pluripotentes/citologia , Animais , Animais Geneticamente Modificados , Proteínas Aviárias/genética , Sequência de Bases , Linhagem Celular , Proliferação de Células , Embrião de Galinha/metabolismo , Galinhas , Quimera/genética , DNA Complementar/genética , Diploide , Transtornos do Desenvolvimento Sexual , Feminino , Células Germinativas , Masculino , Proteínas do Tecido Nervoso/genética , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transplante de Células-TroncoRESUMO
The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell-derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.
