What was the first transgenic organism

When considering the question “what is a transgene and why is it needed,” most scientists would point at the large body of genetic research in use today. Transgenes are an integral part of modern genetic manipulation, but what are they exactly? What is their main underlying purpose and advantages, and what makes them play such a pivotal role in the development of humanized mouse models and various genetic manipulation methods designed to target specific diseases?

What Are Transgenes?

Before going any further: what is a transgene exactly? The concept of a transgene has been broadly understood for more than 100 years, as the advent of genetic research made its way into the 20th century. As time went by, technologies evolved, and transgenes became widely used during the late 1970s and early ‘80s. Transgenes are pieces of genetic material that are used to modify the genome of a certain organism. The modification of the organism’s phenotype is also possible through the use of transgenes. In order to function properly, transgenes require several key components. The promoter is a regulatory sequence that determines where and when the transgene will be activated, while the exon is in charge of protein-coding sequence and the stop sequence. A third element is the bacterial plasmid that is used to deliver these components to the host genome.

“The project was very well managed…in fact, using iTL validated my decision to not try and do this in my own lab. It would have been a catastrophe… (My project manager) was very helpful, always getting back to us in time and explaining every step of the project. I would be glad to serve as a reference for iTL and its staff.”

Claus Fimmel, MD
Loyola University Medical Center

Why Are Transgenes So Important?

When you ask “what is a transgene,” you also have to ask yourself, why were transgenes were developed in the first place? Transgenic organisms have the important role of expressing various genes, which may make them vulnerable to specific disorders or conditions that researchers aim to study. The valuable research gathered from the development of humanized transgenic mice, for example, has been used to develop new treatments for cancer and other, equally debilitating disorders. Advancements in molecular biology allow for new models to be developed, helping scientists study the human genome more closely and conditions such as aging, diabetes, infertility and immune response.

The First Transgenic Organisms

The first transgene to be obtained in controlled conditions through specific, genetic manipulation methods was in 1974. Staphylococcus aureus genes were then introduced successfully into e. coli. The first eukaryotic organism to be used in a gene transfer experiment was yeast in 1978. After yeast, the first transgenic mouse model was developed just one year later. During the first trial experiments, DNA was transferred directly into the target cells via microinjection.

Modern Examples for the Uses of the Transgene

What is a transgene, and how is it used today? Although there are many examples, perhaps the most significant is the use of transgenes in plants and food. Corn, cotton and rapeseed are just a few of the plants that have been genetically modified for the purpose of increasing the yield and the health of crops as much as possible. New mouse models are also being developed on a regular basis, the oncomouse being an entirely new genetically modified mouse species developed for the study of cancer. Considering the issue “what is a transgene and what should we expect in the future,” researchers also point to potential applications like the xenotransplantation of organs, the development of artificial protein-based foods and a cure for fertility-related genetic disorders.

A transgenic animal is one whose genome has been altered by the transfer of a gene or genes from another species or breed.

The photo shows two transgenic mice positioned either side of a plain mouse. The transgenic mice have been genetically modified so that they carry a green fluorescent protein which glows green under blue light. Credit: Ingrid Moen et alet al., BMC Cancer, 12/21 (2012), 1-10.

Connections Frank Ruddle | CRISPR | Monoclonal antibodies | Recombinant DNA

Importance

Transgenic animals are routinely used in the laboratory as models in biomedical research. Over 95 per cent of those used are genetically modified rodents, predominantly mice. They are important tools for researching human disease, being used to understand gene function in the context of disease susceptibility, progression and to determine responses to a therapeutic intervention.Mice have also been genetically modified to naturally produce human antibodies for use as therapeutics. Seven out of the eleven monoclonal antibody drugs approved by the FDA between 2006 and 2011 were derived from transgenic mice.

Transgenic farm animals are also being explored as a means to produce large quantities of complex human proteins for the treatment of human disease. Such therapeutic proteins are currently produced in mammalian cell-based reactors, but this production process is expensive. In 2008, for example, the building of a new cell-based manufacturing facility for one therapeutic protein was estimated to cost over US$500 million. A cheaper option would be to develop a means to produce recombinant proteins in the milk, blood or eggs of transgenic animals. Progress in this area, however, has been slow to-date. Only two biomedical products have so far received regulatory approval. The first is human antithrombin III, a therapeutic protein produced in the milk of transgenic goats, which is used to prevent clots in patients with hereditary antithrombin deficiency receiving surgery or undergoing childbirth. A relatively small herd of goats (about 80) can supply enough human antithrombin III for all of Europe. The second product is a recombinant human C12 esterase inhibitior produced in the milk of transgenic rabbits. This is used to treat hereditary angiodema, a rare genetic disorder which causes blood vessels in the blood to expand and cause skin swellings.

Discovery

The ability to produce transgenic animals is reliant on a number of components. One of the first things needed to generate transgenic animals is the ability to transfer embryos. The first successful transfer of embryos was achieved by Walter Heape in Angora rabbits in 1891. Another important component is the ability to manipulate the embryo. In vitro manipulation of embryos in mice was first reported in the 1940s using a culture system. What is also vital is the ability to manipulate eggs. This was made possible through the efforts of Ralph Brinster, attached to the University of Pennsylvania, who in 1963 devised a reliable system to culture eggs, and that of Teh Ping Lin, based at the California School of Medicine, who in 1966 outlined a technique to micro-inject fertilised mouse eggs which enabled the accurate insertion of foreign DNA.The first genetic modification of animals was reported in 1974 by the virologist Rudolph Jaenisch, then at the Salk Institute, and the mouse embryologist Beatrice Mintz at Fox Chase Cancer Center. They demonstrated the feasibility of modifying genes in mice by injecting the SV40 virus into early-stage mouse embryos. The resulting mice carried the modified gene in all their tissues. In 1976, Jaenisch reported that the Moloney Murine Leukemia Virus could also be passed on to offspring by infecting an embryo. Four years later, in 1980, Jon Gordon and George Scango together with Frank Ruddle, announced the birth of a mouse born with genetic material they had inserted into newly fertilised mouse eggs. By 1981 other scientists had reported the successful implantation of foreign DNA into mice, thereby altering the genetic makeup of the animals. This included Mintz with Tim Stewart and Erwin Wagner at the Fox Chase Cancer Center in Philadelphia; Brinster and Richard Palmiter at the University of Washington, Seattle; and Frank Costantini and Elizabeth Lacy at Oxford University.

Such work laid the basis for the creation of transgenic mice genetically modified to inherit particular forms of cancer. These mice were generated as a laboratory tool to better understand the onset and progression of cancer. The advantage of such mice is that they provide a model which closely mimics the human body. The mice not only provide a means to gain greater insight into cancer but also to test experimental drugs.

Application

Transgenic animals are animals (most commonly mice) that have had a foreign gene deliberately inserted into their genome. Such animals are most commonly created by the microinjection of DNA into the pronuclei of a fertilised egg which is subsequently implanted into the oviduct of a pseudopregnant surrogate mother. This results in the recipient animal giving birth to genetically modified offspring. The progeny are then bred with other transgenic offspring to establish a transgenic line. Transgenic animals can also be created by inserting DNA into embryonic stem cells which are then micro-injected into an embryo which has developed for five or six days after fertilisation, or infecting an embryo with viruses that carry a DNA of interest. This final method is commonly used to manipulate a single gene, in most cases this involves removing or 'knocking out' a target gene. The end result is what is known as a ‘knockout’ animal.Since the mid-1980s transgenic mice have become a key model for investigating disease. Mice are the model of choice not only because there is extensive analysis of its completed genome sequence, but its genome is similar to the human. Moreover, physiologic and behavioural tests performed on mice can be extrapolated directly to human disease. Robust and sophisticated techniques are also easily available for the generic manipulation of mouse cells and embryos. Another advantage of mice is the fact that they have a short reproduction cycle. Other transgenic species, such as pig, sheep and rats are also used, but their use in pharmaceutical research has so far been limited due to technical constraints. Recent technological advances, however, are laying the foundation for wider adoption of the transgenic rat. Transgenic rodents play a number of critical roles in drug discovery and development. Importantly, they enable scientists to study the function of specific genes at the level of the whole organism which has enhanced the study of physiology and disease biology and facilitated the identification of new drug targets. Due to their similarity in physiology and gene function between humans and rodents, transgenic rodents can be developed to mimic human disease. Indeed, an array of transgenic mice models have been produced for this purpose. Mice are being used as models, for example, to study obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, ageing, Alzheimer's disease and Parkinson's disease. They are also used to study different forms of cancer. In addition, transgenic pigs are being investigated as a source of organs for transplants, which if proven clinically safe could overcome some of the severe donor organ shortages. The development of transgenic animals has recently been transformed by the emergence of the new gene editing tool CRISPR which greatly reduced the number of steps involved in the creation of transgenic animals, making the whole process much faster and less costly.

This section on transgenic mice was jointly written by Lara Marks and Dmitriy Myelnikov. For more information see D. Myelnikov, 'Transforming mice: technique and communication in the making of transgenic animals, 1974-1988', unpublished PhD, Cambridge University, 2015.

Transgenic animals: timeline of key events

Smithies was a geneticist and physical biochemist. He first made his mark in 1955 through his invention of starch gel electrophoresis, a technique used to study human protein variation. Later on, in the 1980s he developed a method for targeted gene replacement in mice, now known as gene targeting, for which he was awarded the Nobel Prize for Medicine in 2007. His method facilitated the creation of thousands of lines of mice carrying desired genetic mutations. Such mice are now widely used to investigate the role of many different genes in human health and disease.1925-06-23T00:00:00+0000Founded by Clarence Little, one of the leading researchers into genetic differences governing the rejection of foreign tissues. 1929-01-01T00:00:00+0000Ruddle helped pioneer human gene mapping and established many of the techniques and a framework for setting up the Human Genome Project. He generated, with Jon W. Gordon and George Scango the first successful transgenic mouse. This heralded the development of genetically modified animals as research models to investigate the function of genes and genetic cause of disease. Ruddle also discovered, with William McGinnis, the first human homeobox genes, important regulators of gene development. 1929-08-19T00:00:00+0000Clark was a molecular biologist who used genetic engineering to create the first sheep capable of producing large quantities of human protein. The sheep, Tracy, born in 1990, provided 35g of the alpha-1-antitrypsin in each litre of her milk. The protein is used in the treatment of cystic fibrosis. Clark also managed to develop the first large transgenic animal, a sheep, in which a prion protein gene had been improved. 1951-09-18T00:00:00+00001974-01-01T00:00:00+0000The mice were made with the help recombinant DNA technology. JW Gordon, GA Scangos, DJ Plotkin, J A Barbosa, FH Ruddle, 'Genetic transformation of mouse embryos by microinjection of purified DNA', PNAS USA, 77 (1980), 7380–4.1980-09-01T00:00:00+0000M Capecchi, 'High efficiency transformation by direct microinjection of DNA into cultured mammalian cells', Cell, 22/2 (1980), 479-88.1980-11-01T00:00:00+0000The experiment proved it was possible to transfer a cloned gene into germ-line cells and for the gene to be subsequently transmitted into the offspring. This was the first step towards the development of transgenic mice. F Costantini, E Lacy, 'Introduction of a rabbit b-globin gene into the mouse germ line', Nature, 294 (1981), 92–4.1981-11-05T00:00:00+00001982-12-01T00:00:00+0000The course is started at Cold Harbour Laboratory together with collaborators from other centres.1983-01-01T00:00:00+0000These are created with the objective of studying self-tolerance. 1985-01-01T00:00:00+0000Capecchi, M, 'Site-directed mutagenesis by gene targeting in mouse embyo-derived stem cells', Cell, 51/3 (1987), 503-12.1987-11-06T00:00:00+0000This patent is filed on the basis of work reported in M Brüggeman, HM Caskey, C Teale, H Waldmann, Williams, Surani, and MS Neuberger, A repertoire of monoclonal antibodies with human heavy chains from transgenic mice, Proc Natl Acad Sci USA, 86 (Sept 1989), 6709-13. 1988-01-01T00:00:00+0000USPTO patent 4,736,866 awarded for transgenic mouse with activated oncogenes created by Philip Leder and Timonthy A Stewart at Harvard University. The two scientists isolated a gene that causes cancer in many mammals, including humans, and inserted it into fertilised mouse eggs. The aim was to genetically engineer a mouse as a model for furthering cancer research and the testing of new drugs. It was the first animal ever given patent protection in the USA. 1988-04-12T00:00:00+0000Mouse genetated with genes knocked out that produce the enzyme DNA methyltransfgerase involved in DNA methylation. E. Li, T.H. Bestor, R. Jaenisch, 'Targeted mutation of the DNA methyltransferase gene results in embryonic lethality', Cell, 69/6 (1992), 915-26.1992-06-12T00:00:00+0000Three groups of scientists separately report the successful generation of different strains of transgenic mice for the generation of human monoclonal antibodies. Two of the teams are based in biotechnology companies: GenPharm (led by Nils Lonsberg), Cell Gensys (led by Larry Green) , and the other involved a collaboration (led by Marian Bruggemann and Michael Neuberger) between scientists at the Laboratory of Molecular Biology, Braham Institute and the University of Cologne.1994-01-01T00:00:00+0000Dolly the sheep was created by cloning an adult cell. This was done by transferring the nucleus of an adult sheep's cell to the nucleus of an unfertilised egg cell. It took 277 attempts to achieve success. The work was carried out by Keith Campbell, Ian Wilmut and colleagues at the Rosilin Institute, PPL Therapeutics and the Ministry of Agriculture. 1996-07-05T00:00:00+0000The sheep, Polly, was produced by the same scientists who cloned Dolly the sheep. Polly was one of six cloned lambs which had human genes inserted. The gene that was transferred was linked to then human blood clotting factor IX. Such genetic engineering was done to demonstrate the potential of such recombinant DNA technology combined with animal cloning. It was done in the hope that one day transgenic animals might provide pharmacological and therapeutic proteins and transplant organs to treat human diseases. The work was published in AE Schnieke; et al, 'Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts', Science, 278/5346 (1997), 2130–33.1997-07-09T00:00:00+0000Dolly the sheep was created by cloning an adult cell. This was done by transferring the nucleus of an adult sheep's cell to the nucleus of an unfertilised egg cell. It took 277 attempts to achieve success.2003-02-14T00:00:00+0000Clark was a British molecular biologist who used genetic engineering to create the first sheep capable of producing large quantities of human protein. The sheep, Tracy, born in 1990, provided 35g of the alpha-1-antitrypsin in each litre of her milk. The protein is used in the treatment of cystic fibrosis. Clark also managed to develop the first large transgenic animal, a sheep, in which a prion protein gene had been improved.2004-08-12T00:00:00+0000Panitumumab (Vectibix) was approved by the FDA for the treatment of patients with EGFR-expressing metatastic colorectal cancer. The drug is a fully human monoclonal antibody created with transgenic mice. It was developed by Agensys with Amgen. 2006-09-27T00:00:00+0000The Prize was awarded to to Mario Capecchi, Martin Evans and Oliver Smithies. Their work made it possible to modify specoific genes in the germline of mammals which could produce offspring that carried and expressed the modified gene. Their method is commonly called knockout technology. This has given scientists the means to study the role of specific genes in development, physiology and pathology. 2007-01-01T00:00:00+0000Ruddle helped pioneer human gene mapping and established many of the techniques and a framework for setting up the Human Genome Project. He also generated, with Jon W. Gordon and George Scango the first successful transgenic mouse. This heralded the development of genetically modified animals as research models to investigate the function of genes and genetic cause of disease. Ruddle also discovered, with William McGinnis, the first human homeobox genes, important regulators of gene development. 2013-03-10T00:00:00+0000A pioneer of antibody engineering, Neuberger developed some of the first techniques for the generation of chimeric and humanised antibodies. He also helped create the first transgenic mice for the production of human monoclonal antibodies. His work paved the way for the generation of safer and more effective monoclonal antibody drugs. 2013-10-26T00:00:00+0000The pigs, a small breed known as Bama, had some of their genes disabled. They were developed for use in studying stem cells, gut microbiota, and Laron syndome, a type of dwarfism caused by a mutation in the human GHR gene. The announcement was made at Shenzhen International Biotech Leaders Summit.2015-09-23T00:00:00+0000The aim was to to inactivate 62 endogenous retroviruses in the pig embryos. All pigs have these viruses embedded in their genomes. The presence of such viruses, which can transmit diseases like cancer, is a major hurdle to the transplant of pig organs into humans. The gene editing work was carried out by the geneticist George Church of Harvard Medical School. He and his team presented the results to the US National Academy of Sciences. 2015-10-05T00:00:00+0000Smithies was a British-born American geneticist and physical biochemist. He first made his mark in 1955 through his invention of starch gel electrophoresis, a technique used to study human protein variation. Later on, in the 1980s he developed a method for targeted gene replacement in mice, now known as gene targeting, for which he was awarded the Nobel Prize for Medicine in 2007. His method facilitated the creation of thousands of lines of mice carrying desired genetic mutations. Such mice are now widely used to investigate the role of many different genes in human health and disease. 2017-01-10T00:00:00+0000Collaborative research carried out by scientists at University of Edinburgh, University College London and Imperial College. 2017-04-20T00:00:00+0000A team of scientists managed to engineer mice to express Cas9 and a DNA sequence needed for the gene drive, called a cassette, which encoded a guide RNA that targets a sequence in the TYR gene which affects the mouse coat colour. This provided a means of tracking the frequency of the genetic modification over several generations of mice. The work was published in HA Grunwald et al. 'Super-Mendelian inheritance mediated by CRISPR–Cas9 in the female mouse germline', Nature, January 23, 2019.2019-01-23T00:00:00+0000A team of surgeons led by Robert Montgomery at New York University Langone Health attached the organ to a brain-dead individual who was being maintained on a ventilator. Although the kidney remained outside the body, it worked normally, making urine and creatinine, a waste product. 2021-09-25T00:00:00+0000The transplant was performed on David Bennett, a 57 year old, by doctors at the University of Maryland Medical Center. The heart was taken from a pig that had been genetically modified to knock out several genes that would have led to the organ being rejected by Mr Bennett's immune system. The treatment was considered the last hope for saving Mr Bennett who had heart failure. Mr Bennett was reported to be doing well three days after the operation but died after two months. The team who performed the transplant was led by Muhammad Mohiuddin.2022-01-11T00:00:00+0000

Oliver Smithies was born in Halifax, United Kingdom

Jackson Memorial Laboratories established to develop inbred strains of mice to study the genetics of cancer and other diseases

Frank Ruddle was born in West New York, New Jersey

Anthony J Clark was born in Blackpool, UK

First publication on inserting foreign DNA into mice

Scientists reported the first successful development of transgenic mice

Technique published using fine glass micropipettes to inject DNA directly into the nuclei of cultured mammalian cells. High efficiency of the method enables investigators to generate transgenic mice containing random insertions of exogenous DNA.

First successful transmission of foreign DNA into laboratory mice

Giant mice made with the injection of rat growth hormone

Course started in the molecular embyology of mice

First transgenic mice created with with genes coding for both the heavy and light chain domains in an antibody.

Publication of gene targeting technique for targetting mutations in any gene

Patent application filed for a method to create transgenic mice for the production of human antibodies

First transgenic mouse model created for studying link between DNA methylation and disease

First transgenic mice strains reported for producing human monoclonal antibodies

Dolly the sheep, the first cloned mammal, was born

Birth of first sheep cloned with human genes

Dolly the sheep, the first cloned mammal, died

First fully human monoclonal antibody drug approved

Nobel Prize for Physiology for Medicine awarded for discoveries enabling germline gene modification in mice using embryonic stem cells

Frank Ruddle died in New Haven, Connecticut

Beijing Genomics Institute announced the sale of the first micropigs created with the help of the TALENs gene-editing technique

CRISPR/Cas9 modified 60 genes in pig embryos in first step to create organs suitable for human transplants

Diabetes research using transgenic mice shows the protein P2X7R plays important role in inflammation and immune system offering new avenue for treating kidney disease

CRISPR-Cas9 used to control genetic inheritance in mice

First genetically engineered pig kidney successfully transplanted into a brain-dead human patient

First pig-to-human heart transplant