Genetic engineering / Rekayasa Genetika

Definition

Genetic engineering alters the genetic makeup of an organism using techniques that introduce heritable material prepared outside the organism either directly into the host or into a cell that is then fused or hybridized with the host.[1] This involves using recombinant nucleic acid (DNA or RNA) techniques to form new combinations of heritable genetic material followed by the incorporation of that material either indirectly through a vector system or directly through micro-injection, macro-injection and micro-encapsulation techniques. Genetic engineering does not include traditional animal and plant breeding, in vitro fertilisation, induction of polyploidy, mutagenesis and cell fusion techniques that do not use recombinant nucleic acids or a genetically modified organism in the process. Cloning and stem cell research, although not considered genetic engineering,[2] are closely related and genetic engineering can be used within them.[3] Synthetic biology is an emerging discipline that takes genetic engineering a step further by introducing artificially synthesized genetic material from raw materials into an organism.

If genetic material from another species is added to the host, the resulting organism is called transgenic. If genetic material from the same species or a species that can naturally breed with the host is used the resulting organism is called cisgenic.[5] Genetic engineering can also be used to remove genetic material from the target organism, creating a knock out organism.[6] In Europe genetic modification is synonymous with genetic engineering while within the United States of America it can also refer to conventional breeding methods.[7] Within the scientific community, the term genetic engineering is not commonly used; more specific terms such as transgenic are preferred.

History

Humans have altered the genomes of species for thousands of years through artificial selection and more recently mutagenesis. Genetic engineering as the direct manipulation of DNA by humans outside breeding and mutations has only existed since the 1970s. The term “genetic engineering” was first coined by Jack Williamson in his science fiction novel Dragon’s Island, published in 1951,[8] one year before DNA’s role in heredity was confirmed by Alfred Hershey and Martha Chase,[9] and two years before James Watson and Francis Crick showed that the DNA molecule has a double-helix structure.

In 1972 Paul Berg created the first recombinant DNA molecules by combined DNA from the monkey virus SV40 with that of the lambda virus.[10] In 1973 Herbert Boyer and Stanley Cohen created the first transgenic organism by inserting antibiotic resistance genes into the plasmid of an E. coli bacterium.[11][12] A year later Rudolf Jaenisch created a transgenic mouse by introducing foreign DNA into its embryo, making it the world’s first transgenic animal.[13] In 1976 Genentech, the first genetic engineering company was founded by Herbert Boyer and Robert Swanson and a year later and the company produced a human protein (somatostatin) in E.coli. Genentech announced the production of genetically engineered human insulin in 1978.[14] In 1980, the U.S. Supreme Court in the Diamond v. Chakrabarty case ruled that genetically altered life could be patented.[15] The insulin produced by bacteria, branded humulin, was approved for release by the Food and Drug Administration in 1982.[16]

The first field trials of genetically engineered plants occurred in France and the USA in 1986, tobacco plants were engineered to be resistant to herbicides.[17] The People’s Republic of China was the first country to commercialize transgenic plants, introducing a virus-resistant tobacco in 1992.[18] In 1994 Calgene attained approval to commercially release the Flavr Savr tomato, a tomato engineered to have a longer shelf life.[19] In 1994, the European Union approved tobacco engineered to be resistant to the herbicide bromoxynil, making it the first genetically engineered crop commercialized in Europe.[20] In 1995, Bt Potato was approved safe by the Environmental Protection Agency, making it the first pesticide producing crop to be approved in the USA.[21] In 2009 11 transgenic crops were grown commercially in 25 countries, the largest of which by area grown were the USA, Brazil, Argentina, India, Canada, China, Paraguay and South Africa.[22]

In 2010, scientists at the J. Craig Venter Institute, announced that they had created the first synthetic bacterial genome, and added it to a cell containing no DNA. The resulting bacterium, named Synthia, was the world’s first synthetic life form.[23][24]

Process

Isolating the Gene

Elements of genetic engineering

First, the gene to be inserted into the genetically modified organism must be chosen and isolated. Presently, most genes transferred into plants provide protection against insects or tolerance to herbicides.[25] In animals the majority of genes used are growth hormone genes.[26] Once chosen the genes must be isolated. This typically involves multiplying the gene using polymerase chain reaction (PCR). If the chosen gene or the donor organism’s genome has been well studied it may be present in a genetic library. If the DNA sequence is known, but no copies of the gene are available, it can be artificially synthesized. Once isolated, the gene is inserted into a bacterial plasmid.

Constructs

The gene to be inserted into the genetically modified organism must be combined with other genetic elements in order for it to work properly. The gene can also be modified at this stage for better expression or effectiveness. As well as the gene to be inserted most constructs contain a promoter and terminator region as well as a selectable marker gene. The promoter region initiates transcription of the gene and can be used to control the location and level of gene expression, while the terminator region ends transcription. The selectable marker, which in most cases confers antibiotic resistance to the organism it is expressed in, is needed to determine which cells are transformed with the new gene. The constructs are made using recombinant DNA techniques, such as restriction digests, ligations and molecular cloning.[27]

Gene Targeting

Main article: gene targeting

The most common form of genetic engineering involves inserting new genetic material randomly within the host genome. Other techniques allow new genetic material to be inserted at a specific location in the host genome or generate mutations at desired genomic loci capable of knocking out endogenous genes. The technique of gene targeting uses homologous recombination to target desired changes to a specific endogenous gene. This tends to occur at a relatively low frequency in plants and animals and generally requires the use of selectable markers. The frequency of gene targeting can be greatly enhanced with the use of engineered nucleases such as zinc finger nucleases,[28] [29] engineered homing endonucleases,[30] [31] or nucleases created from TAL effectors.[32] [33] In addition to enhancing gene targeting, engineered nucleases can also be used to introduce mutations at endogenous genes that generate a gene knockout[34] .[35]

Transformation

Main article: Transformation (genetics)

A. tumefaciens attaching itself to a carrot cell

About 1% of bacteria are naturally able to take up foreign DNA but it can also be induced in other bacteria.[36] Stressing the bacteria for example, with a heat shock or an electric shock, can make the cell membrane permeable to DNA that may then incorporate into their genome or exist as extrachromosomal DNA. DNA is generally inserted into animal cells using microinjection, where it can be injected through the cells nuclear envelope directly into the nucleus or through the use of viral vectors. In plants the DNA is generally inserted using Agrobacterium-mediated recombination or biolistics.[37]

In Agrobacterium-mediated recombination the plasmid construct must also contain T-DNA. Agrobacterium naturally inserts DNA from a tumor inducing plasmid into any susceptible plant’s genome it infects, causing crown gall disease. The T-DNA region of this plasmid is responsible for insertion of the DNA. The genes to be inserted are cloned into a binary vector, which contains T-DNA and can be grown in both E. Coli and Agrobacterium. Once the binary vector is constructed the plasmid is transformed into Agrobacterium containing no plasmids and plant cells are infected. The Agrobacterium will then naturally insert the genetic material into the plant cells.[38]

In biolistics particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material will enter the cells and transform them. This method can be used on plants that are not susceptible to Agrobacterium infection and also allows transformation of plant plastids. Another transformation method for plant and animal cells is electroporation. Electroporation involves subjecting the plant or animal cell to an electric shock, which can make the cell membrane permeable to plasmid DNA. In some cases the electroporated cells will incorporate the DNA into their genome. Due to the damage caused to the cells and DNA the transformation efficiency of biolistics and electroporation is lower than agrobacterial mediated transformation and microinjection.[39]

Selection

Not all the organism’s cells will be transformed with the new genetic material; in most cases a selectable marker is used to differentiate transformed from untransformed cells. If a cell has been successfully transformed with the DNA it will also contain the marker gene. By growing the cells in the presence of an antibiotic or chemical that selects or marks the cells expressing that gene it is possible to separate the transgenic events from the non-transgenic. Another method of screening involves using a DNA probe that will only stick to the inserted gene. A number of strategies have been developed that can remove the selectable marker from the mature transgenic plant.[40]

Regeneration

As often only a single cell is transformed with genetic material the organism must be regrown from that single cell. As bacteria consist of a single cell and reproduce clonally regeneration is not necessary. In plants this is accomplished through the use of tissue culture. Each plant species has different requirements for successful regeneration through tissue culture. If successful an adult plant is produced that contains the transgene in every cell. In animals it is necessary to ensure that the inserted DNA is present in the embryonic stem cells. When the offspring is produced they can be screened for the presence of the gene. All offspring from the first generation will be heterozygous for the inserted gene and must be mated together to produce a homozygous animal.

Confirmation

The finding that a recombinant organism contains the inserted genes is not usually sufficient to ensure that the genes will be expressed in an appropriate manner in the intended tissues of the recombinant organism. To examine the presence of the gene, further analysis frequently uses PCR, Southern hybridization, and DNA sequencing, which serve to determine the chromosomal location and copy number of the inserted gene. To examine expression of the trans-gene, an extensive analysis of transcription, RNA processing patterns, and the expression and localization of the protein product(s) is usually necessary, using methods including northern hybridization, quantitative RT-PCR, Western blot, immunofluorescence and phenotypic analysis. When appropriate, the organism’s offspring are studied to confirm that the trans-gene and associated phenotype are stably inherited.

Applications

Genetic engineering has applications in medicine, research, industry and agriculture and can be used on a wide range of plants, animals and micro organism.

Medicine

In medicine genetic engineering has been used to mass-produce insulin, human growth hormones, follistim (for treating infertility), human albumin, monoclonal antibodies, antihemophilic factors, vaccines and many other drugs.[41] Vaccination generally involves injecting weak live, killed or inactivated forms of viruses or their toxins into the person being immunized.[42] Genetically engineered viruses are being developed that can still confer immunity, but lack the infectious sequences.[43] Mouse hybridomas, cells fused together to create monoclonal antibodies, have been humanised through genetic engineering to create human monoclonal antibodies.[44]

Genetic engineering is used to create animal models of human diseases. Genetically modified mice are the most common genetically engineered animal model.[45] They have been used to study and model cancer (the oncomouse), obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging and Parkinson disease.[46] Potential cures can be tested against these mouse models. Also genetically modified pigs have been bred with the aim of increasing the success of pig to human organ transplantation.[47]

Gene therapy is the genetic engineering of humans by replacing defective human genes with functional copies. This can occur in somatic tissue or germline tissue. If the gene is inserted into the germline tissue it can be passed down to that person’s descendants.[48] Gene therapy has been used to treat patients suffering from immune deficiencies (notably Severe combined immunodeficiency) and trials have been carried out on other genetic disorders.[49] The success of gene therapy so far has been limited and a patient (Jesse Gelsinger) has died during a clinical trial testing a new treatment.[50] There are also ethical concerns should the technology be used not just for treatment, but for enhancement, modification or alteration of a human beings’ appearance, adaptability, intelligence, character or behavior.[51] The distinction between cure and enhancement can also be difficult to establish.[52] Transhumanists consider the enhancement of humans desirable.

Research

Knockout mice

Human cells in which some proteins are fused with green fluorescent protein to allow them to be visualised

Genetic engineering is an important tool for natural scientists. Genes and other genetic information from a wide range of organisms are transformed into bacteria for storage and modification, creating genetically modified bacteria in the process. Bacteria are cheap, easy to grow, clonal, multiply quickly, relatively easy to transform and can be stored at -80°C almost indefinitely. Once a gene is isolated it can be stored inside the bacteria providing an unlimited supply for research.

Organisms are genetically engineered to discover the functions of certain genes. This could be the effect on the phenotype of the organism, where the gene is expressed or what other genes it interacts with. These experiments generally involve loss of function, gain of function, tracking and expression.

  • Loss of function experiments, such as in a gene knockout experiment, in which an organism is engineered to lack the activity of one or more genes. A knockout experiment involves the creation and manipulation of a DNA construct in vitro, which, in a simple knockout, consists of a copy of the desired gene, which has been altered such that it is non-functional. Embryonic stem cells incorporate the altered gene, which replaces the already present functional copy. These stem cells are injected into blastocysts, which are implanted into surrogate mothers. This allows the experimenter to analyze the defects caused by this mutation and thereby determine the role of particular genes. It is used especially frequently in developmental biology. Another method, useful in organisms such as Drosophila (fruit fly), is to induce mutations in a large population and then screen the progeny for the desired mutation. A similar process can be used in both plants and prokaryotes.
  • Gain of function experiments, the logical counterpart of knockouts. These are sometimes performed in conjunction with knockout experiments to more finely establish the function of the desired gene. The process is much the same as that in knockout engineering, except that the construct is designed to increase the function of the gene, usually by providing extra copies of the gene or inducing synthesis of the protein more frequently.
  • Tracking experiments, which seek to gain information about the localization and interaction of the desired protein. One way to do this is to replace the wild-type gene with a ‘fusion’ gene, which is a juxtaposition of the wild-type gene with a reporting element such as green fluorescent protein (GFP) that will allow easy visualization of the products of the genetic modification. While this is a useful technique, the manipulation can destroy the function of the gene, creating secondary effects and possibly calling into question the results of the experiment. More sophisticated techniques are now in development that can track protein products without mitigating their function, such as the addition of small sequences that will serve as binding motifs to monoclonal antibodies.
  • Expression studies aim to discover where and when specific proteins are produced. In these experiments, the DNA sequence before the DNA that codes for a protein, known as a gene’s promoter, is reintroduced into an organism with the protein coding region replaced by a reporter gene such as GFP or an enzyme that catalyzes the production of a dye. Thus the time and place where a particular protein is produced can be observed. Expression studies can be taken a step further by altering the promoter to find which pieces are crucial for the proper expression of the gene and are actually bound by transcription factor proteins; this process is known as promoter bashing.

Industrial

By engineering genes into bacterial plasmids it is possible to create a biological factory that can produce proteins and enzymes.[53] Some genes do not work well in bacteria, so yeast, a eukaryote, can also be used.[54] Bacteria and yeast factories have been used to produce medicines such as insulin, human growth hormone, and vaccines, supplements such as tryptophan, aid in the production of food (chymosin in cheese making) and fuels.[55] Other applications involving genetically engineered bacteria being investigated involve making the bacteria perform tasks outside their natural cycle, such as cleaning up oil spills, carbon and other toxic waste.[56]

Agriculture

Bt-toxins present in peanut leaves (bottom image) protect it from extensive damage caused by European corn borer larvae (top image).[57]

One of the best-known and controversial applications of genetic engineering is the creation of genetically modified food. There are three generations of genetically modified crops.[58] First generation crops have been commercialized and most provide protection from insects and/or resistance to herbicides. There are also fungal and virus resistant crops developed or in development.[59][60] They have been developed to make the insect and weed management of crops easier and can indirectly increase crop yield.[61]

The second generation of genetically modified crops being developed aim to directly improve yield by improving salt, cold or drought tolerance and to increase the nutritional value of the crops.[62] The third generation consists of pharmaceutical crops, crops that contain edible vaccines and other drugs.[63] Some agriculturally important animals have been genetically modified with growth hormones to increase their size[64] while others have been engineered to express drugs and other proteins in their milk.[65][66][67]

The genetic engineering of agricultural crops can increase the growth rates and resistance to different diseases caused by pathogens and parasites.[68] This is beneficial as it can greatly increase the production of food sources with the usage of fewer resources that would be required to host the world’s growing populations. These modified crops would also reduce the usage of chemicals, such as fertilizers and pesticides, and therefore decrease the severity and frequency of the damages produced by these chemical pollution.[68]

Ethical and safety concerns have been raised around the use of genetically modified food.[69] A major safety concern relates to the human health implications of eating genetically modified food, in particular whether toxic or allergic reactions could occur.[70] Gene flow into related non-transgenic crops, off target effects on beneficial organisms and the impact on biodiversity are important environmental issues.[71] Ethical concerns involve religious issues, corporate control of the food supply, intellectual property rights and the level of labeling needed on genetically modified products.

Other uses

In materials science, a genetically modified virus has been used to construct a more environmentally friendly lithium-ion battery.[72][73] Some bacteria have been genetically engineered to create black and white photographs[74] while others have potential to be used as sensors by expressing a fluorescent protein under certain environmental conditions.[75] Genetic engineering is also being used to create BioArt[76] and novelty items such as blue roses,[77] and glowing fish.[78]

Opposition and criticism

A 2010 study of Canola found transgenes in 80% of wild (uncultivated or “feral”) varieties in North Dakota, meaning 80% of the plants which had established themselves in the area were genetically engineered varieties. The researchers stated that “we found the highest densities of [such transgene-containing] plants near agricultural fields and along major freeways, but we were also finding plants in the middle of nowhere” adding that “over time,..the build-up of different types of herbicide resistance in feral [natural] canola and closely related weeds, like field mustard, could make it more difficult to manage these plants using herbicides.”

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