Functional transgenesis in laboratory mice was first reported in the early 1980s, when investigators expressed human growth hormone, producing abnormal growth (1). Since that time, innumerable variations in transgene configuration have resulted in greater control over exogenous gene expression levels, timing, tissue/cell type distribution, and gene product localization. Significant progress toward adapting these systems for use in agricultural mammals began a few years after the technology's murine debut, with the production of transgenic rabbits, sheep, pigs (2), goats (3), and cattle (4) expressing exogenous proteins in their milk.
Up to this point, investigators used pronuclear microinjection, the original method developed in mice, to create these transgenic animals. Briefly, fertilized embryos are collected from superovulated donors at the one-cell “zygote” stage before syngamy, the fusion of maternal and paternal pronuclei. Highly purified transgene DNA is injected into one of the two pronuclei, and the injected embryos are allowed to develop in culture. Embryos that survive the process are then transferred into a pseudopregnant recipient uterus for gestation, and live offspring are screened for the presence of the transgene. Although success rates for introducing transgenes in this way vary based on species and transgene DNA construct, they are typically low (usually 1–5% of injected embryos), and other DNA integration strategies, including the use of reverse-transcribed gene transfer in oocytes and intracytoplasmic sperm injection, have been developed to increase efficiency (5).
Somatic cell nuclear transfer (SCNT) is a newer method that has several advantages for genetically cloning valuable transgenic animals, as well as for gene targeting in species for which embryonic stem cells have yet to be isolated, including the ability to perform sequential genetic modifications, targeted DNA insertions, and artificial chromosome transfer using long-term cultured somatic cells. Successful production of live offspring by SCNT was first reported in goats in 1999 (6) and in cattle in 2000 using adult skin fibroblast cells (7). These accomplishments opened new possibilities for biomedical research and for the development of transgenic animals of great value to agriculture and the pharmaceutical industry.
Specifically, the ability to express transgenes in milk-producing animals has resulted in the creation of “bioreactors,” or animals that produce large amounts of a given recombinant protein in their milk, in fully biologically active form through proper posttranslational modification (PTM), for purification and therapeutic use. This approach has been used to produce recombinant tissue plasminogen activator (3, 8), granulocyte colony-stimulating factor (9), Ig (10), and lactoferrin (11) in the milk of goats and cows. A recombinant form of human antithrombin III (ATryn) produced in goats is currently approved in Europe (it has passed clinical trials but has not been approved for use in the U.S.) for the prevention of clotting in surgical patients with hereditary antithrombin deficiency. This is the first instance of a transgenically produced drug being approved for use in humans.
The ability to express transgenes in milk-producing animals has resulted in the creation of “bioreactors.”
In a recent issue of PNAS, Huang et al. (12) describe the results of an ambitious and highly successful project to produce large amounts of a recombinant human enzyme in the milk of transgenic goats, in an effort to develop a preventative treatment for organophosphate (OP) intoxication. The key findings of this study are as follows:
The human butyrylcholinesterase (BChE) gene, driven by a goat β-casein promoter, can be expressed in transgenic goat's milk at concentrations as high as 5 g/liter.
High expression of recombinant BChE (rBChE) was transmitted to offspring through sexual reproduction and by SCNT.
Kilogram quantities of purified, active rBChE were produced—amounts that demonstrate the feasibility of protecting humans against OP poisoning.
PEGylation of rBChE dimers increased plasma half-life in guinea pigs 7-fold.
rBChE protected guinea pigs against lethal doses of OP nerve agents, resulting in no apparent toxicity.
The authors sought to produce large amounts of recombinant rBChE enzyme, which is normally found in human plasma. BChE has some similarities to the neurotransmitter inhibitor acetylcholinesterase (AChE) that allow BChE to act as a so-called “suicide inhibitor” of toxic OP compounds that would normally irreversibly inhibit AChE. Compounds like Sarin, Soman, and VX are neurotoxic because they deplete AChE, leading to unchecked neurological stimulation by the neurotransmitter acetylcholine.
Boosting plasma BChE levels with recombinant protein, the team reasoned, should scavenge OP molecules in the bloodstream and protect against toxicity. However, the 1:1 stoichiometry involved means that a large amount of rBChE is necessary. The authors report the successful development of a transgenic animal-based strategy to produce large quantities of purified rBChE enzyme for use as a blocker of the toxic effects of various compounds, some of which have been used in military and terrorist actions.
Huang et al. (12) first tested their transgenic construct in mice for protein expression, characterization, and PTMs. Next, they extended this strategy to create transgenic goats, followed by herd expansion through natural breeding or cloning (although some clones have different transgene copy numbers, suggesting mosaic expression of the transgene in different somatic cell populations of the founder transgenic animals) and characterization of the protein for activity, PTM, half-life, etc. They showed that transgenic goats produced the active transgene protein in milk in sufficient quantities for prophylaxis of humans at risk for exposure to OP agents.
One of the most striking aspects of this report is how well rBChE worked in an animal model. PEGylated rBChE protected guinea pigs from any apparent toxicity, even after exposure to multiple lethal doses of VX or Soman. Of course, as with any new drug, there is still a long way to go before it will find its way to human patients. For one thing, it remains to be seen whether elevated plasma BChE levels might pose health risks in humans, and any deliberate testing of efficacy in patients is out of the question. Although this treatment clearly has military applications, one can also imagine civilian use to protect personnel in chemical plants, pesticide applicators, and first responders to disasters and terrorist attacks. It will be interesting to see how the standard pharmaceutical regulatory paradigm will adapt to such treatments, which cannot be fully tested for efficacy. In the end, the quest to create such chemical “antiweapons” may serve to make chemical weapons obsolete, or at least less attractive to those who would use them—a goal worthy of greater support and encouragement.
Author contributions: X.Y. and M.G.C. wrote the paper.
The authors declare no conflict of interest.
See companion article on page 13603 in issue 34 of volume 104.
