A new method for producing genetically customized chickens will likely lead to important applications in research as well as in agriculture and the pharmaceutical industry, report scientists at Origen Therapeutics and the University of California, Davis.

The new system uses primordial germ cells (PGCs) -- the very earliest cells that normally mature into sperm and eggs in the chicken embryo -- harvested from the chicken embryo. Marie-Cecile van de Lavoir and her team at Origen Therapeutics demonstrated for the first time that these cells can be isolated, genetically modified and grown indefinitely in the laboratory, while remaining committed to the germ (reproductive) cell lineage. Once inserted into a recipient chicken embryo at the appropriate developmental stage, the modified PGCs will develop normally into sperm and eggs, thus passing on the introduced traits to the next generation of chickens.

The findings from this study were reported in the June 8 issue of the British journal Nature.

"This is the best system available to date for producing transgenic chickens," said Mary Delany, professor and chair of the UC Davis Department of Animal Science, whose team collaborated in the research and demonstrated that the chromosomes of the cultured PGCs were normal. "We anticipate it will have a tremendous impact on developmental biology research, enabling scientists to readily study various genes of interest. The chicken has long been an important model organism for developmental biology, and this makes a great model even better.

"Furthermore, this system has promise for agricultural applications to introduce genes for beneficial traits such as disease resistance and, in the pharmaceutical industry, to introduce genes for human proteins that have therapeutic value," Delany added.

Background

Humans and animals are made up of two general categories of cells: the somatic cells -- sometimes called body cells -- which form tissues and organs, and the germ, or reproductive, cells which form the sperm and eggs (also known as gametes).

Changes that occur, or are induced, in somatic cells are not inherited, but changes in the germ cells are passed on to the next generation. For that reason, the germ or reproductive cells are of keen interest to scientists in the field of developmental biology who study how organisms grow and develop as well as those interested in introducing genetic changes into an organism.

The precursors of the sperm and eggs are called primordial germ cells (PGCs). During development of the embryo -- a process known as embryogenesis -- the PGCs are a distinct lineage from the somatic or body cells, developing at the very earliest stages. When a chicken embryo is about two days old, the PGCs migrate through the bloodstream from one location to the embryo's gonads, the testes or ovaries. There, the PGCs begin differentiation and maturation programs to become functional sperm and eggs, respectively. This developmental program is shared among all vertebrate species.

A new transgenic system

During this study, the Origen team grew PGCs, (twelve male cell lines) that were derived from 114 embryos of barred Plymouth Rock chickens, a breed with black-and-white striped feathers. The researchers analyzed these cells and determined by their cell-surface markers and characteristic morphology that they retained the specific germ-cell features.

Further analysis by Delany's group found that an enzyme called telomerase was present in the cultured cells. Telomerase synthesizes DNA at the end of chromosomes, and its activity provides a genetic mechanism for unlimited cell replication, which is a key characteristic of immortal cell lines. This finding confirmed that the PGCs are capable of replicating long-term when cultured in the laboratory.

To demonstrate that the cells maintained their commitment to become gametes (sperm and eggs), the Origen team injected cultured primordial germ cells into embryos of white-feathered white leghorn chickens. Twenty-four of these embryos, which carried the gametes from the white-feathered breed as well as the injected PGCs from the black-feathered barred Plymouth Rock breed, were raised to adulthood. These birds were mated and produced offspring that carried the black-and-white striped feathers, confirming that the injected cells remained part of the germline, as demonstrated by their ability to pass on this physical characteristic to the next generation.

The Origen team then inserted a gene encoding green fluorescent protein (GFP) into the genome of the cultured PGCs and injected these cells into recipient embryos. When sexually mature, these embryos transmitted the gene encoding GFP to some of their offspring. The gene encoding GFP was expressed in these birds and was inherited by their offspring according to classical Mendelian expectations.

"Collectively, this body of research provides the first demonstration in any species that PGCs can be grown indefinitely in vitro and genetically manipulated, while preserving their ability to contribute to the next generation by producing sperm and eggs," Delany said.

"Dr. Delany brought her internationally recognized expertise in avian cytogenetics to the analysis of PGCs that had been in culture for extended periods" said Robert Etches, vice president of research at Origen Therapeutics. "The combination of in house expertise and world class support from the Delany laboratory allowed Origen to complete the technology quickly and efficiently."

Funding for the study was provided by the U.S. Department of Agriculture and the National Institutes of Health through Small Business Innovation Research grants to Origen Therapeutics and a National Research Initiative grant to UC Davis.

Origen Therapeutics, headquartered in Burlingame, Calif., is a privately held biotechnology company developing product opportunities from their emerging avian transgenic platform. The company's mission is to become a leading developer and producer of complex recombinant protein therapeutics, including human polyclonal antibodies. By taking advantage of the speed and economy with which transgenic chickens can now be produced, Origen is working to establish significant corporate alliances with biotechnology and pharmaceutical companies for the commercialization of its technology.