Genetically modified animals are animals that have beengenetically modified for a variety of purposes including producing drugs, enhancing yields, increasing resistance to disease, etc. The vast majority of genetically modified animals are at the research stage while the number close to entering the market remains small.[1]
The process of genetically engineering mammals is a slow, tedious, and expensive process.[2] As with other genetically modified organisms (GMOs), first genetic engineers must isolate the gene they wish to insert into the host organism. This can be taken from acell containing the gene[3] orartificially synthesised.[4] If the chosen gene or the donor organism'sgenome has been well studied it may already be accessible from agenetic library. Thegene is then combined with other genetic elements, including apromoter andterminator region and usually aselectable marker.[5]
A number of techniques are available forinserting the isolated gene into the host genome. With animalsDNA is generally inserted into usingmicroinjection, where it can be injected through the cell'snuclear envelope directly into thenucleus, or through the use ofviral vectors.[6] The first transgenic animals were produced by injecting viral DNA into embryos and then implanting the embryos in females.[7] It is necessary to ensure that the inserted DNA is present in theembryonic stem cells.[8] The embryo would develop and it would be hoped that some of the genetic material would be incorporated into the reproductive cells. Then researchers would have to wait until the animal reached breeding age and then offspring would be screened for presence of the gene in every cell, usingPCR,Southern hybridization, andDNA sequencing.[9]
Humans havedomesticated animals since around 12,000 BCE, usingselective breeding or artificial selection (as contrasted withnatural selection). The process ofselective breeding, in which organisms with desiredtraits (and thus with the desiredgenes) are used to breed the next generation and organisms lacking the trait are not bred, is a precursor to the modern concept of genetic modification[20]: 1 Various advancements ingenetics allowed humans to directly alter theDNA and therefore genes of organisms. In 1972,Paul Berg created the firstrecombinant DNA molecule when he combined DNA from amonkey virus with that of thelambda virus.[21][22]
In 1974,Rudolf Jaenisch created atransgenic mouse by introducing foreign DNA into itsembryo, making it the world's first transgenic animal.[23][24] However it took another eight years before transgenic mice were developed that passed thetransgene to their offspring.[25][26] Genetically modified mice were created in 1984 that carried clonedoncogenes, predisposing them to developing cancer.[27] Mice with genesknocked out (knockout mouse) were created in 1989. The first transgenic livestock were produced in 1985[28] and the first animal to synthesise transgenic proteins in their milk were mice,[29] engineered to produce human tissueplasminogen activator in 1987.[30]
The first genetically modified animal to be commercialised was theGloFish, aZebra fish with afluorescent gene added that allows it to glow in the dark underultraviolet light.[31] It was released to the US market in 2003.[32] The first genetically modified animal to be approved for food use wasAquAdvantage salmon in 2015.[33] The salmon were transformed with agrowth hormone-regulating gene from aPacific Chinook salmon and a promoter from anocean pout enabling it to grow year-round instead of only during spring and summer.[34]
Somechimeras, like the blotched mouse shown, are created through genetic modification techniques likegene targeting.
GM mammals are created for research purposes, production of industrial or therapeutic products, agricultural uses or improving their health. There is also a market for creating genetically modified pets.[35]
Mammals are the bestmodels for human disease, making genetic engineered ones vital to the discovery and development of cures and treatments for many serious diseases. Knocking out genes responsible forhuman genetic disorders allows researchers to study the mechanism of the disease and to test possible cures.Genetically modified mice have been the most common mammals used inbiomedical research, as they are cheap and easy to manipulate. Examples includehumanized mice created byxenotransplantation of human gene products, so as to be utilized as murinehuman-animal hybrids for gaining relevant insights in thein vivo context for understanding of human-specific physiology and pathologies.[36] Pigs are also a good target, because they have a similar body size, anatomical features,physiology,pathophysiological response, and diet.[37] Nonhuman primates are the most similar model organisms to humans, but there is less public acceptance toward using them as research animals.[38] In 2009, scientists announced that they had successfully transferred a gene into aprimate species (marmosets) and produced a stable line of breeding transgenic primates for the first time.[39][40] Their first research target for these marmosets wasParkinson's disease, but they were also consideringamyotrophic lateral sclerosis andHuntington's disease.[41]
Transgenic pig for cheese production
Human proteins expressed in mammals are more likely to be similar to their natural counterparts than those expressed in plants or microorganisms. Stable expression has been accomplished in sheep, pigs, rats, and other animals. In 2009, the first human biological drug produced from such an animal, agoat, was approved. The drug,ATryn, is ananticoagulant which reduces the probability ofblood clots duringsurgery orchildbirth was extracted from the goat's milk.[42] Humanalpha-1-antitrypsin is another protein that is used in treating humans with this deficiency.[43] Another area is in creating pigs with greater capacity forhuman organ transplants (xenotransplantation). Pigs have been genetically modified so that their organs can no longer carry retroviruses[44] or have modifications to reduce the chance of rejection.[45][46] Pig lungs from genetically modified pigs are being considered for transplantation into humans.[47][48] There is even potential to create chimeric pigs that can carry human organs.[37][49]
Livestock are modified with the intention of improving economically important traits such as growth-rate, quality of meat, milk composition, disease resistance and survival. Animals have been engineered to grow faster, be healthier[50] and resist diseases.[51] Modifications have also improved the wool production of sheep and udder health of cows.[1]
Goats have been genetically engineered to produce milk with strong spiderweb-like silk proteins.[52] The goat gene sequence has been modified, using fresh umbilical cords taken from kids, in order to code for the human enzymelysozyme. Researchers wanted to alter the milk produced by the goats, to contain lysozyme in order to fight off bacteria causingdiarrhea in humans.[53]
Enviropig was a genetically enhanced line ofYorkshire pigs in Canada created with the capability of digesting plantphosphorus more efficiently than conventional Yorkshire pigs.[54][55] The Atransgene construct consisting of apromoter expressed in themurineparotid gland and theEscherichia coliphytase gene was introduced into the pig embryo by pronuclearmicroinjection.[56] This caused the pigs to produce the enzymephytase, which breaks down the indigestible phosphorus, in their saliva.[54][57] As a result, they excrete 30 to 70% less phosphorus in manure depending upon the age and diet.[54][57] The lower concentrations of phosphorus insurface runoff reducesalgal growth, because phosphorus is thelimiting nutrient for algae.[54] Because algae consume large amounts of oxygen, excessive growth can result in dead zones for fish. Funding for the Enviropig program ended in April 2012,[58] and as no new partners were found the pigs were killed.[59] However, the genetic material will be stored at the Canadian Agricultural Genetics Repository Program. In 2006, a pig was engineered to produceomega-3 fatty acids through the expression of aroundworm gene.[60]
In 1990, the world's first transgenicbovine, Herman the Bull, was developed. Herman was genetically engineered by micro-injected embryonic cells with the human gene coding forlactoferrin. TheDutch Parliament changed the law in 1992 to allow Herman to reproduce. Eight calves were born in 1994 and all calves inherited the lactoferrin gene.[61] With subsequent sirings, Herman fathered a total of 83 calves.[62] Dutch law required Herman to beslaughtered at the conclusion of theexperiment. However the Dutch Agriculture Minister at the time,Jozias van Aartsen, granted him a reprieve provided he did not have more offspring after public and scientists rallied to his defence.[62] Together withcloned cows named Holly and Belle, he lived out his retirement atNaturalis, the National Museum of Natural History in Leiden.[62] On 2 April 2004, Herman waseuthanised byveterinarians from theUniversity of Utrecht because he suffered fromosteoarthritis.[63][62] At the time of his death Herman was one of the oldest bulls in the Netherlands.[63] Herman's hide has been preserved and mounted bytaxidermists and is permanently on display in Naturalis. They say that he represents the start of a new era in the way man deals with nature, an icon of scientific progress, and the subsequent public discussion of these issues.[63]
In October 2017, Chinese scientists announced they usedCRISPR gene editing technology to create of a line of pigs with better body temperature regulation, resulting in about 24% less body fat than typical livestock.[64]
Researchers have developed GM dairy cattle to grow without horns (sometimes referred to as "polled") which can cause injuries to farmers and other animals. DNA was taken from the genome ofRed Angus cattle, which is known to suppress horn growth, and inserted into cells taken from an eliteHolstein bull called "Randy". Each of the progeny will be a clone of Randy, but without his horns, and their offspring should also be hornless.[65] In 2011, Chinese scientists generateddairy cows genetically engineered with genes from human beings to produce milk that would be the same as human breast milk.[66] This could potentially benefit mothers who cannot produce breast milk but want their children to have breast milk rather than formula.[67][68] The researchers claim these transgenic cows to be identical to regular cows.[69] Two months later, scientists fromArgentina presented Rosita, a transgenic cow incorporating two human genes, to produce milk with similar properties as human breast milk.[68] In 2012, researchers from New Zealand also developed a genetically engineered cow that produced allergy-free milk.[70]
Scientists have genetically engineered several organisms, including some mammals, to includegreen fluorescent protein (GFP), for research purposes.[73] GFP and other similar reporting genes allow easy visualisation and localisation of the products of the genetic modification.[74] Fluorescent pigs have been bred to study human organ transplants, regenerating ocularphotoreceptor cells, and other topics.[75] In 2011green-fluorescent cats were created to find therapies forHIV/AIDS and other diseases[76] asfeline immunodeficiency virus (FIV) is related to HIV.[77] Researchers from the University of Wyoming have developed a way to incorporate spiders' silk-spinning genes into goats, allowing the researchers to harvest the silk protein from the goats' milk for a variety of applications.[78]
Genetic modification of themyxoma virus has been proposed to conserveEuropean wild rabbits in theIberian peninsula and to help regulate them in Australia. To protect the Iberian species from viral diseases, the myxoma virus was genetically modified to immunize the rabbits, while in Australia the same myxoma virus was genetically modified to lower fertility in the Australian rabbit population.[79] There have also been suggestions that genetic engineering could be used to bring animalsback from extinction. It involves changing the genome of a close living relative to resemble the extinct one and is currently being attempted with thepassenger pigeon.[80] Genes associated with thewoolly mammoth have been added to the genome of anAfrican Elephant, although the lead researcher says he has no intention of using live elephants.[81]
Genetically modified fish are used for scientific research, as pets, and as a food source.Aquaculture is a growing industry, currently providing over half of the consumed fish worldwide.[100] Through genetic engineering, it is possible to increase growth rates, reduce food intake, removeallergenic properties, increase cold tolerance, and provide disease resistance.
Fish can also be used to detect aquatic pollution or function as bioreactors.[101] Several groups have been developingzebrafish to detectpollution by attaching fluorescent proteins to genes activated by the presence of pollutants. The fish will then glow and can be used as environmental sensors.[102][103]
TheGloFish is a brand of genetically modified fluorescentzebrafish with bright red, green, and orange fluorescent color. It was originally developed by one of the groups to detect pollution, but is now part of the ornamental fish trade, becoming the first genetically modified animal to become publicly available as a pet when it was introduced for sale in 2003.[104]
GM fish are widely used in basic research in genetics and development. Two species of fish- zebrafish andmedaka, are most commonly modified, because they have optically clearchorions (membranes in the egg), rapidly develop, and the 1-cell embryo is easy to see and microinject with transgenic DNA.[105] Zebrafish are model organisms for developmental processes,regeneration, genetics, behaviour, disease mechanisms, and toxicity testing.[106] Their transparency allows researchers to observe developmental stages, intestinal functions, and tumour growth.[107][108] The generation of transgenic protocols (whole organism, cell or tissue specific, tagged with reporter genes) has increased the level of information gained by studying these fish.[109]
GM fish have been developed with promoters driving an over-production of "all fish"growth hormone for use in theaquaculture industry, to increase the speed of development and potentially reduce fishing pressure on wild stocks. This has resulted in dramatic growth enhancement in several species, includingsalmon,[110]trout,[111] andtilapia.[112]
AquaBounty Technologies have produced a salmon that can mature in half the time as wild salmon.[113] The fish is an Atlantic salmon with aChinook salmon (Oncorhynchus tshawytscha) gene inserted. This allows the fish to produce growth hormones all year round compared to the wild-type fish that produces the hormone for only part of the year.[114] The fish also has a second gene inserted from the eel-likeocean pout that acts like an "on" switch for the hormone.[114] Pout also haveantifreeze proteins in their blood, which allow the GM salmon to survive near-freezing waters and continue their development.[115] A wild-type salmon takes 24 to 30 months to reach market size (4–6 kg), whereas the producers of the GM salmon say that it requires only 18 months for the GM fish to reach that size.[115][116][117] In November 2015, the FDA of the USA approved theAquAdvantage salmon for commercial production, sale, and consumption,[118] the first non-plant GMO food to be commercialized.[119]
AquaBounty says that to prevent the genetically modified fish from inadvertently breeding with wild salmon, all of the fish will be female and reproductively sterile,[117] although a small percentage of the females may remain fertile.[114] Some opponents of the GM salmon have dubbed it the "Frankenfish".[114][120]
In biological research, transgenic fruit flies (Drosophila melanogaster) aremodel organisms used to study the effects of genetic changes on development.[121] Fruit flies are often preferred over other animals due to their short life cycle and low maintenance requirements. It also has a relatively simple genome compared to manyvertebrates, with typically only one copy of each gene, making phenotypic analysis easy.[122]Drosophila have been used to study genetics and inheritance, embryonic development, learning, behavior, and aging.[123]Transposons (particularly P elements) are well developed inDrosophila and provided an early method to add transgenes to their genome, although this has been taken over by more modern gene-editing techniques.[124]
Due to their significance to human health, scientists are looking at ways to control mosquitoes through genetic engineering. Malaria-resistant mosquitoes have been developed in the laboratory.[125] by inserting a gene that reduces the development of the malaria parasite[126] and then usehoming endonucleases to rapidly spread that gene throughout the male population (known as agene drive).[127] This has been taken further by swapping it for a lethal gene.[128][129] In trials the populations ofAedes aegypti mosquitoes, the single most important carrier of dengue fever and Zika virus, were reduced by between 80% and by 90%.[130][131][129] Another approach is to use thesterile insect technique, whereby males genetically engineered to be sterile out compete viable males, to reduce population numbers.[132]
Otherinsect pests that make attractive targets aremoths.Diamondback moths cause US$4 to $5 billion of damage a year worldwide.[133] The approach is similar to the mosquitoes, where males transformed with a gene that prevents females from reaching maturity will be released.[134] They underwent field trials in 2017.[133] Genetically modified moths have previously been released in field trials.[135] A strain ofpink bollworm that were sterilised with radiation were genetically engineered to express ared fluorescent protein making it easier for researchers to monitor them.[136]
Silkworm, the larvae stage ofBombyx mori, is an economically important insect insericulture. Scientists are developing strategies to enhance silk quality and quantity. There is also potential to use the silk producing machinery to make other valuable proteins.[137] Proteins expressed by silkworms include;human serum albumin,human collagen α-chain, mousemonoclonal antibody andN-glycanase.[138] Silkworms have been created that producespider silk, a stronger but extremely difficult to harvest silk,[139] and even novel silks.[140]
Attempts to produce genetically modified birds began before 1980.[141] Chickens have been genetically modified for a variety of purposes. This includes studyingembryo development,[142] preventing the transmission ofbird flu[143] and providing evolutionary insights usingreverse engineering to recreate dinosaur-like phenotypes.[144] A GM chicken that produces the drugKanuma, an enzyme that treats a rare condition, in its egg passed regulatory approval in 2015.[145]
One potential use of GM birds could be to reduce the spread of avian disease. Researchers atRoslin Institute have produced a strain of GM chickens (Gallus gallus domesticus) that does not transmitavian flu to other birds; however, these birds are still susceptible to contracting it. The genetic modification is anRNA molecule that prevents the virus reproduction by mimicking the region of the flu virus genome that controls replication. It is referred to as a "decoy" because it diverts the flu virus enzyme, thepolymerase, from functions that are required for virus replication.[146]
A team of geneticists led byUniversity of Montana paleontologistJack Horner is seeking to modify a chicken to express several features present in ancestralmaniraptorans but absent in modern birds, such as teeth and a long tail,[147] creating what has been dubbed a 'chickenosaurus'.[148] Parallel projects have produced chicken embryos expressing dinosaur-like skull,[149] leg,[144] and foot[150] anatomy.
Gene editing is one possible tool in the laying hen breeding industry to provide an alternative toChick culling. With this technology, breeding hens are given a genetic marker that is only passed down to male offspring. These males can then be identified during incubation and removed from the egg supply, so that only females hatch. For example, the Israeli startup eggXYt usesCRISPR to give male eggs a biomarker that makes then glow under certain conditions.[151] Importantly, the resulting laying hen and the eggs it producers are not themselves genetically edited. The European Union's Director General for Health and Food Safety has confirmed that made in this way eggs can be marketed,[152] although none are commercially available as of June 2023.[153]
The first experiments that successfully developedtransgenic amphibians into embryos began in the 1980s withXenopus laevis.[154] Later, germline transgenic axolotls inAmbystoma mexicanum were produced in 2006 using a technique called I-SceI-mediated transgenesis which utilizes the I-SceI endonucleaseenzyme that can break DNA at specific sites and allow for foreign DNA to be inserted into the genome.[155] BothXenopus laevis and Ambystoma mexicanum are model organisms used to studyregeneration. In addition, transgenic lines have been produced in othersalamanders including the Japanese newt Pyrrhogaster andPleurodeles watl.[156] Genetically modified frogs, in particularXenopus laevis andXenopus tropicalis, are used indevelopment biology. GM frogs can also be used as pollution sensors, especially forendocrine disrupting chemicals.[157] There are proposals to use genetic engineering to controlcane toads in Australia.[158][159] Many lines of transgenic X. laevis are used to study immunology to address how bacteria and viruses cause infectious disease at the University of Rochester Medical Center's X. laevis Research Resource for Immunobiology (XLRRI).[160] Amphibians can also be used to study and validate regenerativesignaling pathways such as theWnt pathway.[161][160] The wound-healing abilities of amphibians have many practical applications and can potentially provide a foundation for scar-free repair in human plastic surgery, such as treating the skin of burn patients.[162]
Amphibians like X. laevis are suitable for experimentalembryology because they have large embryos that can be easily manipulated and observed during development.[163] In experiments with axolotls, mutants with white pigmented skin are often used because their semi-transparent skin provides an efficient visualization and tracking method forfluorescently tagged proteins likeGFP.[155] Amphibians are not always ideal when it comes to the resources required to produce genetically modified animals; along with the one to two-year generation time,Xenopus laevis can be considered less than ideal for transgenic experiments because of itspseudotetraploid genome.[163] Due to the same genes appearing in the genome multiple times, the chance ofmutagenesis experiments working is lower.[164] Current methods of freezing and thawing axolotl sperm render them nonfunctional, meaning transgenic lines must be maintained in a facility and this can get quite costly.[155][165] Producing transgenic axolotls has many challenges due to their large genome size.[165] Current methods of generating transgenic axolotls are limited to random integration of the transgenecassette into the genome, which can lead to uneven expression or silencing.[156] Gene duplicates also complicate efforts to generate efficientgene knockouts.[165]
Despite the costs, axolotls have unique regenerative abilities and ultimately provide useful information in understanding tissue regeneration because they can regenerate their limbs, spinal cord, skin, heart, lungs, and other organs.[165][166] Naturally occurring mutant axolotls like the white strain that are often used in research have a transcriptional mutation at the Edn3 gene locus.[167] Unlike other model organisms, the first fluorescently labeled cells in axolotls were differentiated muscle cells instead of embryos. In these initial experiments in the early 2000s, scientists were able to visualize muscle cell regeneration in the axolotl tail using a microinjecting technique, but cells could not be traced for the entire course of regeneration due to too harsh conditions that caused early cell death in labeled cells.[156][168] Though the process of producing transgenic axolotls was a challenge, scientists were able to label cells for longer durations using a plasmid transfection technique, which involves injecting DNA into cells using an electrical pulse in a process calledelectroporation.Transfecting axolotl cells is thought to be more difficult because of the composition of theextracellular matrix (ECM). This technique allows spinal cord cells to be labeled and is very important in studying limb regeneration in many other cells; it has been used to study the role of the immune system in regeneration. Usinggene knockout approaches, scientists can target specific regions of DNA using techniques likeCRISPR/Cas9 to understand the function of certain genes based on the absence of the gene of interest. For example, gene knockouts of theSox2 gene confirm this region's role in neural stem cell amplification in the axolotl. The technology to do more complex conditional gene knockouts, or conditional knockouts that give the scientist spatiotemporal control of the gene is not yet suitable for axolotls.[165] However, research in this field continues to develop and is made easier by recent sequencing of the genome and resources created for scientists, including data portals that contain axolotl genome and transcriptome reference assemblies to identifyorthologs.[169][170]
ThenematodeCaenorhabditis elegans is one of the major model organisms for researchingmolecular biology.[171]RNA interference (RNAi) was discovered inC. elegans[172] and could be induced by simply feeding them bacteria modified to expressdouble stranded RNA.[173] It is also relatively easy to produce stable transgenic nematodes and this along with RNAi are the major tools used in studying their genes.[174] The most common use of transgenic nematodes has been studying gene expression and localisation by attaching reporter genes. Transgenes can also be combined with RNAi to rescue phenotypes, altered to study gene function, imaged in real time as the cells develop or used to control expression for different tissues or developmental stages.[174] Transgenic nematodes have been used to study viruses,[175] toxicology,[176] and diseases[177][178] and to detect environmental pollutants.[179]
Systems have been developed to create transgenic organisms in a wide variety of other animals. The gene responsible foralbinism insea cucumbers has been found, and used to engineerwhite sea cucumbers, a rare delicacy. The technology also opens the way to investigate the genes responsible for some of the cucumbers more unusual traits, includinghibernating in summer,eviscerating their intestines, and dissolving their bodies upon death.[180]Flatworms have the ability to regenerate themselves from a single cell.[181][182] Until 2017 there was no effective way to transform them, which hampered research. By using microinjection and radiation, scientists have now created the first genetically modified flatworms.[183] Thebristle worm, a marineannelid, has been modified. It is of interest due to its reproductive cycle being synchronized with lunar phases, regeneration capacity and slow evolution rate.[184]Cnidaria such asHydra and the sea anemoneNematostella vectensis are attractive model organisms to study theevolution ofimmunity and certain developmental processes.[185] Other organisms that have been genetically modified includesnails,[186]geckos,turtles,[187]crayfish,oysters,shrimp,clams,abalone,[188] andsponges.[189]
Food products derived from genetically modified (GM) animals have not yet entered the European market. Nonetheless, the on-going discussion about GM crops [1], and the developing debate about the safety and ethics of foods and pharmaceutical products produced by both GM animals and plants, have provoked varying views across different sectors of society[190]
Genetic modification andgenome editing hold potential for the future, but decisions regarding the use of these technologies must be based not only on what is possible, but also on what is ethically reasonable. Principles such as animal integrity, naturalness, risk identification and animal welfare are examples of ethically important factors that must be taken into consideration, and they also influence public perception and regulatory decisions by authorities.[191]
The utility of extrapolating animal data to humans has been questioned. This has led ethical committees to adopt the principles of the four Rs (Reduction, Refinement, Replacement, and Responsibility) as a guide for decision-making regardinganimal experimentation. However, complete abandonment oflaboratory animals has not yet been possible, and further research is needed to develop a roadmap for robust alternatives before their use can be fully discontinued.[192]
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