Barnyard Biotechnology May Soon Produce New Medical Therapeutics

by Vernon G. Pursel, Caird E. Rexroad, Jr., and Robert J. Wall, Research Physiologists, Gene Evaluation and Mapping Laboratory, Livestock and Poultry Sciences Institute, ARS, USDA, Beltsville, MD.

Farm animals may soon play an important role in providing new lifesaving medical products for treating a variety of human diseases. This is certainly not a new role for farm animals. Thousands of people have benefited from biomedical products derived from farm animals. Notable examples include replacement heart valves, insulin to treat diabetes, and oxytocin to induce labor during childbirth.

However, a number of biologically important hormones, enzymes, blood coagulation factors, and immunological agents that are vital to medical therapy are sufficiently different among species that nonhuman sources are simply not effective. In some cases, these rare proteins can be isolated from human blood and tissues, but this isolation procedure is extremely expensive. Also, these materials' limited availability does not meet their demand, and they carry the risk Of transmitting infectious diseases such as hepatitis B and AIDS.

In recent years, the genetic codes of a number of these medically important proteins have been deciphered. Using recombinant DNA techniques, copies of the human genes responsible for encoding these proteins can now be transferred into micro-organisms or mammalian cells. In many cases, micro-organisms and cells "transformed" in this manner can read the genetic code of the human gene and produce the desired protein. Some of the less complex hormones, such as growth hormone, are being commercially produced in micro-organisms. Although some of the more complex proteins can be successfully synthesized by mammalian cells, maintaining living mammalian cells in large quantities in the laboratory is technically difficult and extremely expensive and, in many instances, even cultivated mammalian cells cannot faithfully produce the desired product.

A potentially more cost-effective means of producing human pharmaceuticals is to actually produce them in farm animals. This can be achieved by introducing a copy of the gene for the human pharmaceutical into the farm animal's genome. Animals that contain copies of these genes are called "transgenic animals." Genetically engineered farm animals would be a particularly attractive production system if the gene could be engineered so that the pharmaceutical is secreted into blood, milk, urine, or tissue. This would allow for easy "harvest" of the product.

Gene Transfer Methods

The primary method used to produce transgenic animals is actually quite simple, in principle. Copies of the cloned genes are transferred into embryos through a finely drawn glass capillary tube inserted into the pro-nucleus of a recently fertilized egg. Microinjection is done under a microscope with the aid of special optics and a micromanipulator to hold the egg and guide the insertion of the tube. After the injection, the eggs are transferred into the oviduct of a foster mother to develop.

For reasons still unknown, only a low percentage of treated eggs incorporate a transgene into one of the chromosomes. However, genes have now been successfully transferred into cattle, goats, pigs, and sheep. In farm animals, about 10 percent of these injected embryos result in a birth, and about 10 percent of the newborn are transgenic. Only about half of these transgenic animals have transgenes that function properly (express the transgene). When these transgenic animals mature and produce offspring,usually about half of them will inherit the transgene.

Targeting Gene Expression to Specific Tissues

To obtain human proteins from transgenic livestock, the DNA sequences encoding the chosen protein must be linked to the promoter/regulatory DNA sequences known to function specifically in the desired tissue. Although some of the necessary information about how promoter/regulatory DNA functions has already been established through fundamental research with mice, considerable research on gene regulation in farm animals is essential because genetic controls differ among species. For example, when a growth hormone gene was transferred into mice, only low levels of expression were detected, whereas when the same gene was transferred into sheep, extraordinarily high concentrations of growth hormone were produced. To further complicate matters, microinjected genes seem to integrate into chromosomes randomly. This is a problem because it appears that nearby genes can influence the function of the transgene, and consequently the level of gene expression can vary greatly from one transgenic animal to the next.

Mammary Gland as Bioreactor. Many scientists consider synthesis of human proteins in animals' mammary glands to be the most promising alternative to production of proteins in tissue culture. Milk proteins are synthesized by the mammary gland and secreted into milk in large quantities.

The average protein content of milk in dairy cattle and goats is 3.1 percent, while sheep and pig milk average 6.8 and 5.9 percent protein, respectively. A number of the milk proteins are synthesized exclusively in the mammary epithelial cells. Thus, use of promoter/regulatory sequences from the genes for these milk proteins will confine expression of human proteins to mammary glands and should avoid exposing the animal's whole body to the human proteins.

In addition, use of the mammary gland for biosynthesis of human proteins offers the advantages of simplicity for collection of large volumes of raw materials on a continual basis, a relatively low maintenance cost of the four-legged bioreactor after the initial transgenic animals are produced, and the ability to produce numerous progeny if the demand for the therapeutic protein should require it.