Genetically modified animals

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Compared to plants, the genetic engineering of animals is sometimes much more complex. This is particularly true for mammals , as the egg cell is not directly accessible and methods of in vitro fertilization must therefore be used.

Methods

In mammals, the methodology has evolved in recent years from the random integration of DNA by microinjection to precise genetic microsurgery ( genome editing ).

Microinjection

The original method involves injecting the gene into the fertilized egg cell ( zygote ), in whose genome the injected DNA is integrated at a random location. In mammals, the method is very time-consuming, as the injected eggs have to be introduced into wet nurses by embryo transfer in order to enable normal development. This technique, often referred to as microinjection , is very well established in mice. In the case of larger mammals such as sheep, goats, pigs and cattle, the technology is complex, since a large breeding program has to be provided in order to have enough eggs and foster animals available. Microinjection into fertilized egg cells is relatively inefficient and only allows the addition of a gene whose activity can hardly be predicted since the random location of integration in the genome essentially determines gene activity.

Embryonic stem cells

A targeted gene modification is possible in mammals if the genetic manipulation is carried out in embryonic stem cells . Here , a gene can be inserted or changed site-specifically, ie at a defined point in the genome, by means of homologous recombination . This process, which is quite inefficient, has been greatly simplified by genome editing . After successful genetic manipulation of the embryonic stem cells, these are integrated into blastocysts . The developing embryo is a chimera , ie it contains not only normal cells but also transgenic cells that are randomly integrated into the various tissues and organs during the development of the embryo. Those animals are then selected in which the transgenic cells are involved in the development of the germ line so that the transgene can be passed on to the following generations through the germ cells .

Somatic cell nuclear transfer

Since it has so far not been possible to obtain embryonic stem cells from farm animals, cloning is used in these animals . Here, the cell nucleus is taken from a genetically modified cell with the desired properties and injected into the enucleated egg cell. The developing embryo is transferred to a wet nurse to create a transgenic animal. The method known as somatic cell nuclear transfer is called, English somatic cell nuclear transfer (SCNT) is relatively inefficient because it often leads to malformations. The key to success is the cell line that serves as the core donor. It must not have any changes in the genome that make normal development impossible. In principle, a specific gene modification with genome editing can also take place in the donor cell line . However, the effort is very great, since cells which are suitable for cloning often only have a low rate of homologous recombination. Based on the published data, which is based on more than 30,000 implanted embryos from cattle, pigs, goats and sheep, the yield is around 1% of genetically modified animals.

Genome editing

One method of choice is genome editing directly in the fertilized egg, as this approach results in a good yield of developing embryos (approx. 50%) and around 10% to 40% of the newborns carry the desired knock-out . Obviously, genome editing in the egg is so efficient that both alleles are often mutated. Genome editing makes it possible to introduce a desired mutation that occurs in a certain animal breed into another animal breed. This makes it possible to create a new breed of animal that only contains the desired mutation. Thus the gene pool of the animal species concerned is not changed. In principle, a corresponding animal can also be obtained by breeding . However, this process is very complex because it has to be carried out over many generations. In addition, you will always have other genes in the newly established breed that may be undesirable. In the last five years after the genome editing method was developed, more than 300 experiments have been successfully carried out on cattle, pigs, sheep and goats.

Three different methods are being developed to introduce genetic changes into farm animals with high efficiency using genome editing.

Electroporation of the zygote

With the method of electroporation , the reagents for genome editing are introduced into the zygote , the fertilized egg cell, by means of short electrical pulses . The egg cell is then cultured into a blastocyst in a test tube and then implanted in the uterus of a mother animal so that the embryo can continue to develop until it is born. This procedure is much simpler than microinjection and somatic cell nuclear transfer .

Transduction with adeno-associated viruses

The gene constructs that are to be introduced into the zygote are introduced into the DNA of adeno-associated viruses . The zygote is infected with these recombinant viruses and is then transferred to the mother animal as a blastocyst in exactly the same way as after electroporation . This approach has so far been used with great success in mice, but it has not yet been used in farm animals.

Surrogate Sire Technology

The Surrogate Sire (surrogate father) technology uses male animals that cannot produce sperm because they do not have spermatogonal stem cells . These sterile sires are transformed into fertile animals by introducing spermatogonial stem cells into the testes. Since the spermatogonal stem cells can be modified with genome editing, animals with the desired genetic properties can be established. This new approach has only been successful in mice so far.

Laboratory and experimental animals

For basic research, genetically modified animals are an important approach to recording the functioning of the various genes in the entire organism. These experimental animals , especially mice, also serve to develop and optimize methods of gene modification.

Pets

Pets are usually kept by humans for joy, adornment or as companions. The genetic engineering allows these animals, often through domestication were adapted to humans, in addition to the wishes of the people to change.

Fluorescent zebrafish

Under the brand name " GloFish ", green fluorescent fish species for aquarium enthusiasts have been sold in the USA. These genetically modified fish are not permitted in the European Union.

Miniature pig

The Chinese research institute BGI has inactivated the gene for the growth hormone receptor in the mini pig breed Bama through genome editing with the " Transcription Activator-like Effector Nuclease " . These genetically modified pigs weigh only 15 kg instead of the normal 35 to 50 kg and were initially planned as experimental animals that allow an inexpensive infrastructure. They were then also offered as pets.

Farm animals

Humans have kept livestock for thousands of years , breeding specific animals that are particularly suitable for their needs. Since the breeding of farm animals is very complex, it is hoped that through the use of genetic engineering, new breeds will be obtained that are useful for humans. Either non-species genes ( transgenes ) are used or specific gene changes are inserted through genome editing .

Increased productivity and reduced environmental impact

Fast growing salmon

A genetically modified salmon was the first food approved by the FDA in the USA in November 2015 . This genetically modified salmon (AquAdvantage salmon) contains an additional growth hormone, so that it is ready for slaughter after 18 months and not only after 3 years. In order to rule out outcrossing with wild forms of salmon, only triploid female animals that are sterile are raised. In addition, the rearing is carried out on land, whereby the escape into the sea is prevented by several barriers.

A US appeals court revoked the FDA approval of the Canadian product in January 2016, pending legal regulation for labeling the product for consumers.

In 2017, 4.5 tons of genetically modified salmon were freely sold in Canada. This is the first and so far only example of a genetically modified livestock that has been released for human consumption.

Pigs with faster growth and reduced environmental pollution

The inefficient feed conversion in pig fattening leads to pollution of the environment with phosphate and nitrate . The phosphate load is due in particular to the fact that pigs cannot digest the phytate from the vegetable diet. In order to enable the utilization of the phytate, transgenic pigs were bred as early as 2001, which produce a bacterial phytate-splitting enzyme (phytase) in the saliva . In these transgenic pigs, up to 75% reduced excretion of phosphate was observed. Based on this finding, transgenic pigs were produced in 2018, in which three bacterial enzymes are also secreted in the saliva that break down certain polysaccharides of plant cells, so that 20% less nitrate is excreted. The improved feed conversion led to a 23% increase in the growth rate of the transgenic pigs.

Increased muscle mass

In cattle, sheep and goats, there are breeds with greater muscle mass. In many cases, this property can be attributed to a mutation that leads to a reduced production of myostatin . Since myostatin inhibits muscle formation, mutations in the myostatin gene lead to increased muscle mass, which leads to an improvement in meat production. Since many breeds of farm animals want to develop a lot of muscle mass, there is great interest in crossing this property. This is very complex with classical breeding, so that genome editing is an attractive approach. In a convincing way, the CRISPR / Cas method was used to knock out the myostatin gene in sheep . 53 blastocysts that arose from injected egg cells were implanted in foster animals and gave birth to 22 lambs, of which eight mutations were present in both alleles . All of these sheep with a homozygous mutation are healthy and have increased muscle mass. The researchers argue that with this approach, for example, merino sheep with high-quality wool could also be turned into a good meat supplier. A logical continuation of this approach is the introduction of the mutation in Texel sheep, which is responsible for the high meat quality. In this case the mutation is a single base change outside of the protein coding sequence normally found in sheep.

Dehorning of cattle

The dehorning of cattle is a common practice, since the horns of a risk of injury among the animals, but also represent for the owner. This dehorning is done by destroying the horn system in calves with a branding iron and is a painful procedure. Some cattle breeds, for example Aberdeen Angus , are polled and molecular genetic analyzes have shown that a mutation in the POLLED gene is responsible for this. In order to introduce polled cattle into proven dairy cattle breeds such as Holstein , very complex breeding programs are necessary in order to obtain the desired characteristics for a good milk production in the polled offspring. As an alternative, the mutation in the POLLED gene was introduced into cells of horned Holstein cattle through genome editing with a transcription activator-like effector nuclease (TALEN) . With the help of somatic cell nuclear transfer , these cells were then used to raise cattle that carry the mutation in the POLLED gene. From the transfer of 295 blastocysts into foster animals, two healthy polled cattle were obtained, which have all the typical characteristics of Holstein cattle. It has thus been possible to genetically modify a proven dairy cattle breed so that it does not have horns and still has all the properties for a high milk yield.

Improved health

Virus resistant pigs

Resistance to foot and mouth disease

Foot and mouth disease is a highly contagious viral disease that can also affect pigs. To suppress the replication of the foot-and-mouth disease virus , a team of Chinese scientists created transgenic pigs that produce a small RNA that, through RNA interference, suppresses the production of the viral envelope protein VP1, which is essential for the virus to replicate in cells is. It is currently unclear whether this protection is effective against all seven subtypes of the virus and whether variants of the virus do not appear relatively quickly, the genome of which is changed by mutation in such a way that the RNA interference can no longer work.

Resistance to epidemic late abortion

Epidemic late abortion in pigs (porcine reproductive and respiratory syndrome, PRRS) is triggered by the PRRS virus and leads to high losses in pig herds. The CD163 protein, which is necessary for the penetration of the PRRS virus, was mutated through genome editing with the CRISPR / Cas system . These genome-edited animals are completely normal, but resistant to infection with the PRRS virus. Whether this resistance works against all subtypes of the PRRS virus has not yet been investigated.

Resistance to African swine fever

The African swine fever is a usually fatal viral disease of domestic pig , but against the Warthog , is tolerant, an African wild form. This difference is based on three DNA sequence differences that lead to corresponding amino acid exchanges in the RELA gene. RELA is a subunit of the transcription factor NF-κB , which is involved in the regulation of the immune response . By genome editing was the Roslin Institute , the RELA gene of the domestic pig mutated so in Scotland that it codes for the form, as present in the warthog. Direct evidence that these genome-edited domestic pigs are tolerant of African swine fever is still pending.

Removal of retroviruses

Organ transplants in humans can only be carried out to a limited extent because only a few human donor organs are available. A possible use of organs from animals ( xenotransplantation ) is therefore being discussed, with the pig being particularly suitable as a donor. A problem with this approach is that porcine retroviruses (PERV) , which are integrated into the pig genome , could be transmitted to human cells after transplantation from pig cells. In order to rule out this possibility, an international research team inactivated all endogenous retroviruses with the help of the CRISPR / Cas method and thus established a pig breeding line that could be used for xenotransplanations.

Mastitis Resistant Cows

Mastitis , the inflammation of the mammary gland, in cows causes great economic damage in agriculture. A Chinese research team has genome editing using a zinc finger nuclease that specifically the beta casein - locus recognizes the human lysozyme integrated gene in this locus. Since this gene locus is active in the mammary gland, an approximately 100-fold increased concentration of lysozyme was found in the milk of these transgenic cows. This increased amount of lysozyme, as well as the fact that human lysozyme is 10 times more active, explains that the transgenic cows have a high resistance to bacteria that cause mastitis.

Combating avian influenza

Avian influenza (avian influenza) is a very contagious disease of chickens and other poultry that can also be transmitted to humans. To combat this disease, transgenic chickens have been developed in which the virus is prevented from multiplying . For this purpose, the chickens were equipped with an expression cassette , which produces a piece of RNA that serves as bait for the viral polymerase . Instead of binding to the virus genome and thereby helping the virus to replicate , the polymerase clings to this bait. The transgenic chickens still die of avian influenza, but no longer infect other chickens. The aim is the complete immunization of chickens against the influenza A virus H5N1 . It is still unclear whether these transgenic chickens and their eggs can be placed on the market.

Improved nutritional value

Milk with increased lactoferrin and lysozyme

The antimicrobial proteins lactoferrin and lysozyme are found in human milk in 10 to 100 times higher concentrations than in the milk of farm animals. In order to increase the content of these proteins, transgenic cows and goats have been bred to contain human lactoferrin or lysozyme in amounts of their milk equivalent to that of humans. So far, no farm animals with human proteins in their milk have been approved for commercial use.

Farm animals as models for biomedical research

Animal models are important for studying genetically determined diseases in humans in order to recognize misdirected signaling pathways and to identify options for their treatment. Since mice and rats are often unsuitable, more and more farm animals, especially pigs and sheep, are used, the anatomy , physiology , lifespan and size of which correspond better to humans. The mutations identified in humans are inserted into the farm animals using genetic engineering methods, in particular genome editing . So far, the focus has been on cystic fibrosis , diabetes mellitus , cardiovascular diseases, cardiac arrhythmias , cancer and many other diseases, the origin of which in humans is often based on genetic defects. To what extent these models also result in concrete improvements in therapy can only be seen in the future.

Manufacture of therapeutically important substances

As early as 1991, attempts were made to produce human lactoferrin in transgenic cows in order to research the possibilities of producing therapeutically important substances. The corresponding bull Herman caused a sensation at the time. This basic research resulted in a recombinant human C1 esterase inhibitor (rhC1-INH, Conestat alfa ) in 2010 , which is produced in the milk of transgenic rabbits and is marketed by Pharming for the treatment of hereditary angioedema (trade name Ruconest ). Previously, in 2006, recombinant human antithrombin (rhAT) was the first substance obtained from transgenic animals to be approved for therapeutic use (prevention of venous thromboembolism in surgical interventions). As a result, further recombinant proteins were isolated from the milk of transgenic rabbits and from the protein of transgenic chickens. Both systems allow the production of pharmaceutically important proteins, the essential modifications of which, in particular glycosylations , only take place in animal cells. So far, however, they have all been niche products, as they can only be used for the treatment of rare diseases, such as Wolman's disease .

insects

Mosquitoes and cabbage moths with a death gene

The yellow fever mosquito ( Aedes aegypti ), which has been spread worldwide by humans, transmits the viruses of yellow fever , dengue fever and Zika fever through its bites . Since no vaccines against dengue and Zika fever have yet been developed, direct control of the mosquito is very important. The use of insecticides is not optimal, as the effect also affects many beneficial insects and the mosquitoes have also become partially resistant to the insecticides used. Therefore, one tries to contain the populations of these mosquitoes with biological pest control . The company Oxitec used genetic engineering to breed yellow fever mosquitoes that carry a deadly gene whose activity can be blocked in the laboratory by tetracycline. When the males of these genetically modified mosquitoes are released, they mate with the normal females so that the death gene is passed on to the offspring. Since no tetracycline is available in the wild population, all offspring that carry this death gene will die. Field tests on the Cayman Islands, Panama and Brazil have shown a reduction in the number of yellow fever mosquitoes by 80 to 95%. It is still unclear whether this reduction will be sufficient to significantly combat the Zika epidemic in South America. In particular, it is a very big challenge to breed the correspondingly high number of genetically modified males of mosquitoes. Similar attempts have been made to control the cabbage moth , whose caterpillars cause considerable damage to cabbage species.

Malaria mosquitoes with resistance genes

Malaria is caused by unicellular Plasmodium parasites that are transmitted by mosquitoes of the genus Anopheles . With no malaria vaccine currently available, fighting the Anopheles is an important approach. Since the use of insecticides is problematic, approaches are also being tested in which the mosquitoes are genetically modified. Particularly interesting is the approach in which resistance genes are introduced into mosquitoes in order to block the reproduction of plasmodium in the mosquito. For this purpose, recombinant antibodies against proteins of Plasmodium falciparum , the main pathogen causing malaria in humans, were introduced into Anopheles. These transgenic mosquitoes prevent the malaria pathogens from multiplying in the mosquito. In order to efficiently introduce these resistance genes into wild populations of Anopheles, the Gene Drive procedure was used, in which the introduced gene spreads from one allele to the second, so that after being released by outcrossing in the first generation, around 99% of the offspring in both Alleles that carry resistance genes. Release experiments have not been carried out as it is unclear whether such experiments could get out of control.

Economic and ethical aspects

Economic aspects

Since practically no animals modified by genetic engineering are currently kept for commercial purposes, a concrete balance is currently not possible. The commercially available pharmaceutical products only play a very minor role.

Animal welfare

Genetically modified animals should meet the criteria of animal welfare . In certain cases, such as dehorning by genome editing , it is argued that this is associated with less stress for the animal, since the mechanical dehorning of the young animals is painful. Improved health is also for the benefit of the animal.

Risks to the ecosystem

The risk that genetically modified livestock will cross with their wild forms is very unlikely. An important exception are certainly transgenic fish, such as genetically modified salmon , which may therefore only be bred under strict controls.

Whether the genetic modification of wild animals, such as the introduction of resistance genes against malaria in mosquitoes, is ethically justifiable is currently the subject of intense debate. It is unclear to what extent the ecosystem could be irreversibly changed. This is especially true if, with Gene Drive, genetic changes occur in both alleles in the first generation and an unstoppable change could occur. Proponents of this technique argue that spontaneous mutagenic processes change the gene drive system in the long term in such a way that the spread would be stopped. The use of Gene Drive to eliminate invasive animals is certainly premature at the moment and requires a worldwide agreement.

Individual evidence

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