Genetic engineering is a more focused form of the selective breeding that humans have been performing for thousands of years. While selective breeding focuses on breeding two organisms with desirable phenotypes together, genetic engineering focuses on directly manipulating an organism's DNA to change its phenotype. Selective breeding changes the DNA indirectly whereas genetic engineering directly changes the DNA of an organism.
What is genetic engineering?
Genetic engineering, as previously mentioned, builds upon the selective breeding that humans have been using for thousands of years to reinforce desirable phenotypical characteristics within organisms. By breeding organisms with the desired characteristic together past humans hoped to reinforce this phenotype or create new ones.
Some classic examples of selective breeding include the numerous dog breeds and modern grains we have today, which evolved from wild wolves and grasses respectively.
To learn more about this first form of indirect genetic engineering read our article Selective Breeding!
Instead of breeding organisms together and hoping the desired outcome occurs, genetic engineering takes influencing phenotypes to another level by making the desired changes directly to the DNA (genotypes). These changes are performed through a variety of techniques, mostly involving the use of recombinant DNA technology. The organisms that result from this genetic manipulation are known as genetically modified organisms (GMOs).
Recombinant DNA technology is a very important research tool in biology. It consists of several laboratory techniques that allow scientists to manipulate, isolate and combine different DNA fragments of interest to create genes with new functions and produce desirable proteins. This so-called recombinant DNA is then usually transferred into a bacterial or yeast cell where it is copied and expressed.
The Genetic Engineering Process
The process of genetically modifying an organism or a cell involves several key steps, each of which may be achieved through a variety of means. However, whatever the mechanism used the end goal is the same.
Selection of a Target Gene
Genetic engineering begins with the identification of the genes the scientist wishes to insert into the genetically modified organism. Which genes are required depends on what we want the organism to do. Where the trait is controlled by a single gene only one gene needs to be moved or modified. This is the simplest way to insert a new trait into a genetically modified organism. An example of this is the insertion of the insulin gene into Escherichia coli for the production of human insulin.
Many phenotypical traits are however polygenic or the result of the action of many genes. These include a large number of metabolic pathways, amongst other traits. These are more complicated to use in genetic engineering as each gene responsible for the traits, or at least enough of them to produce the desired part of the trait, must be identified.
Genes may be identified using either forward or reverse genetics. Forward genetics identifies the desired phenotype first, then finds the gene or genes responsible for it. By inserting DNA sequences into the genome at known locations and looking for changes in the phenotype, allowing the phenotype to be linked to that gene. Reverse genetics functions the opposite way. Thanks to modern genetics techniques, large amounts of sequence data are often available early on in the research process. This allows for the identification of genes early, which may later be linked to phenotypes through introducing mutations or a variety of other means.
Manipulation of the Desired Gene
Once the desired gene is identified it must be extracted from the organism ready to be inserted into another. The gene must then be manipulated in order to ready it for insertion into the genetically modified organism that is to be created. This involves the modification of the gene to allow it to be expressed by the organism it is to be inserted into.
Gene Extraction
The gene must be extracted from the cell in order to be further manipulated. Although in vivo modification of DNA is possible, it's also harder. The cell is first ruptured in order to release the contents into the solution. This is done either mechanically, such as by grinding or even freezing or chemically breaking the plasma membrane. The DNA is then purified, with the necessary steps depending on the lysis method used, ready for downstream uses.
Gene Isolation
In order to insert the gene into another organism, it must be separated from the DNA that makes up the rest of the source organism's genome. How this is performed is dependent on whether the sequence of the gene is known or not. The most widespread technique uses restriction enzymes to isolate the gene and other proteins to insert it into a vector like a bacterial plasmid or a virus. This process is known as molecular cloning!
Restriction enzymes are very important tools for genetic engineering and recombinant DNA technology. These bacterial proteins cut DNA from all organisms at a sequence-specific site, leaving DNA fragments with known sequence ends. This allows scientists to manipulate and insert these DNA fragments easily. There are over three thousand of these enzymes each recognising and cutting a specific DNA site!
Vectors are molecular biology tools and DNA molecules (usually bacterial or viral) used to facilitate the transfer of other DNA into a host cell. The vectors are the vehicles in molecular cloning! They also usually help with expressing and replicating the exogenous DNA in the host.
Gene Insertion
In order for the genetically modified organism to express the desired gene, the now modified gene must be either inserted into its genome or as extrachromosomal DNA. The transmission of the modified gene to offspring can be controlled by controlling where the gene is inserted. Insertion into somatic cells means that the gene cannot be passed on, but insertion into germline cells allows it to be passed down.
Before the gene can be inserted into its expression system it must be modified to allow efficient transcription and translation of the gene. Generally, some form of marker is added to allow for the identification of successfully modified organisms, such as antibiotic resistance genes or fluorescent proteins. A promoter region that encourages the transcription of the gene is also usually added in order to increase the expression of the desired phenotype. A terminator region is used to instruct the expression system to terminate transcription.
There are several techniques used to physically force the insertion of external DNA into a host cell. Each technique has different specificities. The most famous techniques include:
- Transformation - Insertion of DNA in bacteria using recombinant DNA plasmids.
- Transduction - Recombinant viral vectors are used to insert genes into mammalian cells.
- Transfection - Insertion of small molecules like DNA or RNA into eukaryotic cells via non-viral means.
Biolistic transformation is an unusual type of transformation where nucleic acids are shot into organisms in high-speed particle bombardment! Heavy metal particles are coated in the gene, and then literally fired into the cell using a gene gun, carrying the gene with them.
There are several types of transfection methodologies. It may be done via physical means, which force the genetic material into the cell, or by introducing pores (electroporation) into the membrane through which DNA may enter. Chemicals may also be used to trigger the entry of the genetic material into the cell, for example in lipofection.
Targeting of Gene Insertion
Most techniques used to genetically modify organisms insert the desired gene at random places within the genetic material of a cell. While this may work fine and have no negative effects, it may also result in disruption of genes within the organism and consequently their function, which can affect the cell. More recently, programmable enzymes have been developed to allow precise genetic modifications. These enzymes known as nucleases create double-strand breaks within the DNA at specific points, allowing DNA repair mechanisms to be hijacked to insert DNA at that site. These nuclease systems include TALENs, zinc finger nucleases (ZFNs) but especially CRISPR/Cas which is revolutionizing genetic engineering and the world!
CRISPR/Cas stands for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated protein (Cas). CRISPR/Cas is an RNA/protein complex that was discovered as part of the bacterial immune response against viral infection. Its current fame stems from the fact that it's the ultimate molecular scissors! CRISPR/Cas is essentially the best gene-editing tool available, allowing us to precisely introduce base-specific changes in DNA.
Uses and Examples of Genetic Engineering
Genetic engineering has many uses both in and out of the laboratory in many fields such as agriculture, research, industry, medicine, novelty items, and conservation.
Genetic Engineering in Medicine
In medicine, genetic engineering has been used for a variety of purposes. One of the main examples is the production of drugs, such as the bacterial production of human insulin, which was the first example of the use of bacteria to mass-produce human biological molecules. Currently, these techniques are used to create many things, including monoclonal antibodies, hormones, vaccines, coagulation factors and other proteins.
Fig. 1 - A diagram showing the production of insulin through genetic engineering
By modifying animals to have certain genetic diseases, the underlying causes of these diseases can be studied and cures tested. Modifying animal genetics may also remedy the shortage of organs for transplantation by increasing the compatibility of animal organs during xenotransplantation. While genetic engineering may be used to allow cures to be tested, it may also be used as a curative in itself. Gene therapy is used to genetically modify humans to correct genetic diseases such as sickle cell anaemia, Duchenne's Muscular Dystrophy (DMD) and leukaemia.
Fig. 2 - The genetic modification of a goat
Gene therapies are genetic engineering techniques that use genes to directly treat or cure an inherited or acquired genetic disorder. With the advent of the CRISPR/Cas, gene-editing based gene therapy is becoming increasingly popular and successful at directly correcting base-specific disease-causing mutations.
Genetic Engineering in Research
Genetically engineered organisms may be used in research. By inserting genes into bacteria, a near-infinite supply of them may be easily and cheaply provided. Other avenues of research centre around modifying the function of genes within an organism to study the role and functions of the gene. The modification may involve alterations to the function of the gene, tracking of the protein the gene encodes or analysis of the gene expression.
Genetic Engineering for Novelty Products
Genetic engineering has also been used to produce several novelty products, which usually stem from rebranded organisms that were created as part of research projects.
One such example are GloFish, which are fish genetically engineered to express fluorescent proteins in a range of colours. They were initially developed in a lab at the National University of Singapore as part of a research project aiming to create a fish that could detect environmental pollutants and fluoresce. Other examples of novelty genetically modified organisms include bacteria modified to be able to create art, glowing plants and new coloured flowers.
Figure 3. Glofish, a novelty use of genetic engineering with roots in serious research.
Genetic Engineering in Industry
Genetically modified organisms are used in industry to create large amounts of desired proteins, in a similar manner to how they are used to create many medications. They have also been used in waste management, including feeding on oil, plastics, extracting heavy metals and other contaminants from land and many other uses. They have also been used to create materials including carbon nanotubes and lithium batteries. They are also used in the production of food and as sensors for numerous environmental factors.
Genetic Engineering in Agriculture
The use of genetically modified organisms in agriculture is a very controversial topic, however, it provides many opportunities to solve large issues facing society. Plants have been modified to become more resistant to pathogens, be more nutritious, have larger yields, produce different compounds, grow faster or better resist environmental conditions.
Genetic Engineering in Conservation
Genetic engineering has been used in conservation to allow plants to resist pathogens, create viruses that can control invasive species or vaccinate native organisms. It could also potentially be used to help native organisms adapt to climate change and other threats.
Benefits and Risks of Genetic Engineering
There are a number of advantages and disadvantages of genetic engineering, which are surmised below.
Advantages of Genetic Engineering
- It allows organisms to be modified to do exactly what scientists need.
- It solves reliance on other potentially more destructive technologies, like pesticides.
- It allows the production of substances otherwise difficult, expensive or impossible to manufacture in large volumes.
Disadvantages of Genetic Engineering
- The genetic modification may "escape" and enter other organisms. Predicting the long-term consequences of this is very difficult.
- The modification of organisms may have unintended consequences for the organism.
- It raises several ethical and moral issues, such as can you patent life, and should we "play god".
Genetic Engineering - Key takeaways
- Genetic engineering allows for direct changes to be made to the DNA.
- This allows the enhancement of already occurring phenotypes or the addition of new ones to an organism.
- The gene or genes responsible for the phenotype must first be identified then extracted and modified, before being inserted into the desired organism.
- Genetic engineering may be used for medicine, research, novelty, industry, agriculture, conservation and many other areas.
- Genetic engineering is a versatile technique; however, it could have many unintended side effects.
References
- Figure 3: Glofish (https://commons.wikimedia.org/wiki/File:Glofish_shark_purple.jpg) by Ccm272. Licensed by CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0/deed.en).
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