Institute of Science in Society
Foods derived from genetically modified animals are likely to be contaminated by potent vaccines, immune regulators, and growth hormones, as well as nucleic acids, viruses, and bacteria that have the potential to create pathogens and to trigger cancer
Prof. Joe Cummins and Dr. Mae-Wan Ho
Heritable versus non-heritable modifications
The Codex Alimentarius Commission of the United Nations is preparing guidelines for safety assessment of foods derived from recombinant-DNA animals , which is a sure sign that GM animal food is coming to our table.
Codex distinguishes between heritable and non-heritable genetic modification of food animals. Heritable genetic modification involves genetic changes that persist in sperm and egg while non-heritable modification involves the introduction of modified genes such as vaccines into the somatic tissue of animals. Codex asks: “Are there specific food safety questions (e.g. with regard to types of vectors) that should be considered relative to the assessment of safety of food from animals containing heritable versus non-heritable traits?”
We present an overview of heritable and non-heritable modifications, which are not as distinct as Codex thinks, and point to risks that have not been seriously considered. This article is base on a report we submitted to Codex , Genetically Modified Food Animals Coming , which contains all the detailed references.
Heritable alteration or genetic modification (GM) of food animals has been achieved since the early 1980s, mostly by injecting naked DNA. Between 1 and 20 million copies of the transgene (gene to be integrated into the animal genome) are injected into the embryo pronucleus (the nucleus before fertilization) or into the egg cytoplasm, with at most about one percent of injected embryos becoming transgenic animals. The transgenes integrate randomly, though rare instances of homologous recombination with host genes may occur.
A number of different vectors have been used to deliver transgenes in animals. Transposons (mobile genetic units capable of transferring genes) are not widely used in vertebrates. Lentivirus (lenti-, Latin for “slow”), a genus of slow viruses of the Retroviridae family characterized by a long incubation period, can deliver a significant amount of genetic information into the DNA of the host cell, and are among the most efficient gene delivery vectors. HIV (human immunodeficiency virus), SIV (simian immunodeficiency virus), and FIV (feline immunodeficiency virus) are all examples of lentiviruses that have been used successfully with farm animals such as chicken, pig and cow. They are about 50 times more efficient than DNA injection at producing transgenic animals. One problem encountered is that the long terminal repeats of the integration vector interfere with the inserted gene's promoter. Homologous recombination has been used to produce specific gene “knock outs” by replacing an active gene with an inactive one. “Knock in” refers to the integration of a foreign gene at a specific target, disrupting the target gene by inserting the transgene.
Transgenes are designed according to rules that result in gene expression in the host animal, such as the presence of at least one intron, exclusion of GC rich regions, particularly CpG rich motifs. Gene sequences called insulators are often included; these contain transcription enhancers and enhancer blockers to avoid cross talk with adjacent genes, and chromosome openers that modify histones to allow the transcription machinery to be expressed. Finally, RNAi may be used to inactivate specific genes either as heritable transgenes or as non-heritable gene treatments. A vector based on HIV dramatically increased the efficiency of producing transgenic animals, thereby greatly reducing cost. Foetal fibroblast cells can be modified and then cloned to produce transgenic animals.
A novel approach was to transfect germ cell tissue in neonatal testis by electroporation, which was then grafted onto the backs of nude mice (nude mice are immune deficient and tolerate grafts from mammalian tissues). The nude mice, previously castrated, produced mature transgenic sperm that functioned well in in vitro fertilization to produce transgenic farm animals. The technique has been used successfully in cattle, pigs and even humans (though without producing an actual human as yet). The technique is promoted for humans as a means of allowing men requiring irradiation cancer treatment to set aside viable sperm for in vitro fertilization .
‘Improving' the nutritional value and health benefits of livestock
Transgenic clones of cattle producing milk with higher levels of beta casein and kappa casein proteins were created to improve emulsion, processing and heat stability. Rare natural forms of the caseins were used to transform embryonic fibroblasts, with as many as 84 copies of the genes integrated randomly in the genome, no doubt causing huge disruption. The fibroblasts were then used to produce clones of the cattle. Nine cows expressing the transgenes produced milk with up to 20 percent increase in beta-casein and double the level of kappa-casein. The overall health of the transgenic cattle was not discussed, let alone the health impacts of the milk used as food.
This is just one example in a whole range of genetically modified ‘neutraceuticals', animals and animal products that are supposed to provide enhanced nutritional value.
Cloned transgenic pigs have been produced rich in beneficial omega-3 fatty acids normally obtained by eating fish. The transgene consisted of a synthetic n-3 fatty acid desaturase from the roundworm C. elegans driven by an aggressive cytomegalovirus enhancer and chicken beta-actin promoter, accompanied by a selection marker gene for neomycin resistance. Such constructs are typical in attempts to make the transgenic animals over-express the gene product. Pig foetal fibroblasts were transformed and then used to clone transgenic pigs. The transgenic pigs produced high levels of omega-3 fatty acids and a significantly reduced ratio of n-6/n-3 fatty acids. As before, the overall health of the cloned transgenic pigs was not extensively discussed, nor the health impacts of the transgenic pig used as food.
Recombinant human protein C was expressed in the milk of cloned transgenic pigs, also created by transforming foetal pig fibroblasts. Human protein C is an anti-coagulant found in the blood, and serves as a therapy for many disease states. The transgenic pigs produced the therapeutic protein, which protected the pigs against blood clot, but with a risk of pulmonary embolism.
Pigs expressing an E. coli salivary phytase produced low phosphorus manure. Phytase increases the availability of feed phosphorous and decreases its release in manure, thereby eliminating environmental pollution by phosphorus.
Transgenic chickens expressing bacterial beta-galactosidase hydrolyze lactose in the intestine, using it as an energy source, which would have caused diarrhoea to normal chickens. Early chicken embryos were transformed using the spleen necrosis retrovirus vector (SNTZ) . SNTZ is an avian immunosuppressive retrovirus that infects non-replicating cells, not only of birds but of some mammals as well. It has an extraordinarily high mutation rate, and that is not a defect in the replication-deficient vector.
Transgenic fish are poised for commercial release. These will either be produced in confined land-locked ponds, fish pens in confined fjords or sounds, or released to open seas or lakes. Landlocked ponds provide protection from environmental release while fish pens are notoriously unreliable and tend to harbour sea lice or other parasites and pathogens. It would seem most prudent to limit production of transgenic fish, if at all, to landlocked ponds, to avoid or reduce the potentially deleterious impact of transgenic fish on the general environment.
Fish genes are most frequently used in producing transgenic fish, but it would be a mistake to regard the transgenic fish “substantially equivalent” to the native fish, as even the Codex consultation document acknowledges that, “transgenic expression of non-native proteins in plants may lead to structural variants possessing altered immunogenicity.”
AquaBounty Inc. first applied to the US FDA (Food and Drug Administration) in 1999 to release a transgenic Atlantic salmon. The transgenic Atlantic salmon contains a Chinook salmon growth hormone gene driven by the ocean pout antifreeze promoter, resulting in a dramatic increase in growth rate. AquaBounty announces that it is also developing fast growing strains of fin fish known as AquAdvantage™ fish, capable of reducing growth to maturity time by as much as 50 percent. It is expecting FDA approval in 2006 and c ommercial launch in 2009. Scientists have expressed concerns over the release of sexually reproducing transgenic fish; realistic models show that it can lead to the extinction of both the natural and the transgenic population . AquaBounty has produced triploid transgenic Atlantic salmon supposed to be 100 percent sterile; however, the sterility may be “leaky”, and indeed some fertile animals have been produced  ( Floating Transgenic Fish in a Leaky Triploid Craft ) .
Transgenic Coho salmon, carp, tilapia and mud loach are all in the pipelines. The transgenic mud loach grew 35 times faster than the wild type fish, resulting in giant mud loaches that were ready for market after only 30 days.
Transgenic zebra fish have been sold in United States pet shops since 2003  ( Transgenic Fish Coming ). The transgenic zebra fish were projected to be capable of over-wintering in US southern and south-western waters. FDA allowed the release of the zebra fish because the animals did not fall into their jurisdiction. As the animals have been released, their presence in the natural environment should be monitored as a model for the release of transgenic food fish.
Non-heritable modifications of food animals include a number of applications such as DNA vaccination, transgenic probiotic bacteria as vector for vaccines and growth hormones, using RNAi (RNA interference) for epigenetic modifications, and stem cell chimeric animals whose somatic tissue but not the germ cells are transgenic. Non- heritable alterations are taking place or being implemented without full review of the impact on food and the environment, mainly because they do not fall under the rubric of genetic modification.
Naked DNA vaccines
It has been shown since the 1990s that ingested foreign DNA survives transiently in the gastrointestinal tract and enters the bloodstream of mice. Since then, naked DNA has found many applications, especially as DNA vaccines. DNA vaccines can be applied by a variety of routes including intradermal, intravenous, intramuscular, intraperitoneal, subcutaneous, sublinqual, intravaginal, intrarectal, via internasal inhalation, intranasal instillation, ocular and biolistic delivery. Gene vaccines are becoming commonplace and have the advantage of raising antibodies to a target antigen specifically. However, DNA immunization can stimulate florid local inflammation. DNA vaccines are commonly delivered in polyethyenimine complexes, where the plasmid DNA remains active in cells at least 12 days after injection.
DNA vaccines are used in both farm animals and fish, and there has been no study on whether there is any carry over of the vaccine DNA into food prepared from vaccinated animals.
DNA vaccines have been created against pork tapeworms in pigs, bovine herpes virus 1 in cattle, and mastitis caused by Staphylococcus aureus in cows.
A recombinant plasmid DNA vaccine was made to control infectious bursal disease of young chickens characterized by immunosuppression and mortality generally at 3 to 6 weeks of age..
A recombinant plasmid DNA vaccine was prepared to control viral hemorrhagic septicemia, a systemic infection of various salmonid and a few non-salmonid fishes caused by a rhabdovirus (a single stranded RNA virus). A DNA vaccine was made to protect against Mycobacterium marinum that causes tuberculosis in fish and shellfish and cutaneous lesions in humans.