Something's Happening at the Zoo: Cloned Goats Join Sheep, Mice, and Cows
Dolly and Cumula, move over. In the May issue of Nature Biotechnology, three cloned goats are introduced to the world by researchers at Tufts University School of Veterinary Medicine (Grafton, MA) and Genzyme Transgenics Corp. (Framingham, MA) in a paper entitled "Production of goats by somatic cell nuclear transfer." These as yet unnamed goats are not just what are quickly becoming garden-variety clones, they are transgenic goats as well, carriers of the human antithrombin III (rhAT) gene. And what makes this report particularly compelling is the fact that one of the goats produced significant quantities of rhAT in her milk. According to Dr. Yann Echelard of Genzyme Transgenics, this is the first medically significant molecule to be produced in a cloned animal, putting this system in the forefront of pharming models.
How did they do it? A variety of cell types—adult, embryonic, differentiated, and non-differentiated—have been employed in the cloning exercises to date. In this work, the choice was a fetal cell line, derived from fetal cells of a cross between a founder male goat carrying the rhAT gene and a female nontransgenic. Completing this scenario is the presence in the transgene construct of a hormone responsive element that targets the protein to mammary cells (goat ß-casein-rhAT cDNA transgene).
These researchers also altered the usual protocol for producing clones in several respects, by using activated oocytes as recipients for the donor somatic cells, and by serum stimulating donor cells that had been previously deprived of serum for a number of days. Several methods of nuclear implantation were employed to maximize success, two of which were productive—oocytes arrested at metaphase II and calcium-activated telophase II oocytes. A third method, ethanol-induced calcium-activated telophase II oocytes, while giving rise to good cleavage rates among the embryos, did not produce any viable offspring.
Of some 38 recipient does (combining the data from the three methods of preparing oocytes) two carried pregnancies to term, leading to the birth of three goats (one single goat and a set of twins). Several techniques were used to characterize the genotype of the offspring. Southern blotting and hybridization to a human antithrombin III probe revealed the presence of the transgene in the DNA of the cloned animals. Fluorescent in situ hybridization showed that all three contained the inserted DNA on chromosome 5, identical to the donor cells, and RFLP analysis of a highly polymorphic histocompatability locus showed that all three goats were the same and identical to the donor cell line.
"Pharmers": Take note of this work. For one thing, goats are attractive systems for recombinant protein production because with their high productivity, an easily manageable herd can give rise to significant amounts of protein. Furthermore, there are fewer health risks associated proteins isolated from goats—scrapie is virtually unheard of in goats. And while the success of this method for producing transgenic animals is roughly the same range as that of micro-injection (2–3% range), mosaicism, seen frequently with transgenics, was not observed in the genotype of the clones.
For more information: Dr. Yann Echelard, Genzyme Transgenics Corp., One Mountain Rd., Framingham, MA 01701-9322. Tel: 508-620-9700. Fax: 508-370-3797. Or contact: Dr. Eric W. Overstrom, Departments of Biomedical Science and Anatomy and Cellular Biology, Tufts University School of Veterinary Medicine, 200 Westboro Rd., North Grafton, MA 01536. Tel: 508-839-7940. Fax: 508-839-7091.
By Laura DeFrancesco