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Methods and Applications of Genetic Engineering

Genetic Engineering Applications

Hormone, Enzyme, and Vaccine Production

Bioengineering is used to create organisms that act as living manufacturing facilities. Hormones, proteins, and vaccines for human use have been widely produced by using modified microorganisms.

Potentially useful genes are acquired using restriction endonucleases, enzymes that cut deoxyribonucleic acid (DNA), or by polymerase chain reaction (PCR), which selectively copies the target DNA many times. In both cases the gene is removed from its source organism, cloned or replicated, and inserted into a vector that mediates transfer into an organism that will express the gene, typically bacteria or yeast. The bacteria or yeast with the new gene is then grown in large quantities, producing the gene product while they grow and reproduce. The gene product is then separated and purified for use. Using bacteria or yeast is beneficial because of the low cost to grow and keep these organisms, the minimal space required to grow them, and their short generation times. There are several DNA sequences readily available that function well in bacteria and yeast to alter the relative expression level of engineered genes. Altering these sequences leads to increasing the expression level of the gene, called upregulation, or decreasing the expression level, called downregulation.

The earliest commercial use of genetic technologies was the mass production of hormones, such as somatostatin and insulin. In the 1960s and 1970s researchers, such as American geneticists Herbert Boyer and Stanley Cohen, discovered that pieces of DNA from different organisms could be combined to produce recombinant DNA, artificially constructed DNA from multiple origins. Soon after it was discovered that this recombinant DNA could be inserted back into an organism where it functioned properly. Many biological products are harvested from natural sources that are in short supply, or there are ethical drawbacks associated with harvesting them. Applied applications of recombinant DNA were rapidly developed to produce these types of products industrially. Insulin was originally harvested from the pancreases of pigs or cows. In 1982, the company Eli Lilly produced the first human treatment using recombinant DNA, the production of human insulin in Escherichia coli (E. coli) bacteria. The human insulin gene was removed with restriction endonucleases and placed into a plasmid that was cloned into E. coli. The bacteria were left to grow, where they produced thousands of insulin molecules that could be purified for medicinal use.

Insulin Synthesis Using E. coli

Some people are unable to produce or regulate insulin, an important hormone in the body that helps to control the use and storage of glucose. Insulin can now be synthesized using recombinant DNA in E. coli and extracted to provide lifesaving medicine for people in need.
There are now many examples of useful products that are produced using recombinant DNA and associated genetic engineering technologies. Vaccines for animal or human use, hormones for animal or human use, and enzymes used in food or beverage processing are all regularly produced using recombinant DNA technologies in bacteria or yeast cells. For example, the chemical erythropoietin, a signalling molecule that stimulates red blood cell production, is low in anemic humans and its production with recombinant DNA technology has provided a therapeutic solution for patients. Similarly, human growth hormone produced with recombinant DNA provides a useful treatment for children with growth disorders.

Several agricultural crops (canola, corn, cotton, soybean) have been produced with recombinant DNA that have a gene from a bacteria, Bacillus thuringiensis, that provides protection against herbivorous insects in the order Lepidoptera. The production of cheese requires an enzyme called chymosin that aids in the coagulation of milk. Until the 1990's, this enzyme was harvested from the fourth stomach of calves, where it aided in the digestion of their mother's milk. The chymosin gene in calves was cloned and inserted into plasmid vectors that delivered the recombinant DNA to a yeast host. This yeast not only produces chymosin when grown, it excretes it into its environment where it is easily separated for purification.

Gene Therapy

Gene therapy attempts to correct a genetic disorder by transplanting normal genes into cells to replace missing or malfunctioning cells.

Genes, wholly or in part, determine the course of many diseases, such as hemophilia and Huntington's disease. Genetic technology and genetic engineering can be used as treatments for diseases. Gene therapy is the use of recombinant DNA technology to modify a person's genes to combat a disease. In general, altering the expression of a gene, repairing a gene that is malfunctioning, or replacing a malfunctioning gene with a functional copy in the context of disease treatment all fall under the category of gene therapy.

The first treatments receiving approval in the United States were in 2017 to treat non-Hodgkin lymphoma in adults and acute lymphoblastic leukemia in adolescents. Treatments for a heritable condition leading to vision loss and for individuals with hemophilia A were also approved in the U.S. in 2017. Only certain conditions are amenable to gene therapy. If the disease is caused by one well understood gene, and the biology of the resulting disease is well known, it is a good candidate for gene therapy. Gene therapy is less well suited for diseases stemming from more than a few genes or where there is little known about the underlying mechanism of the disease. There are still many technical complications before gene therapy can be widely applied. Transferring new genes or pieces of genes into living human beings in sufficient quantity and specificity is technically challenging and has many ethical restrictions. Gene therapy has the potential for misuse. Since it is now possible to edit genomes, it is possible to cause unintended complications and consequences during the pursuit of therapies. The ethical implications of gene therapy are still being discussed and must be fully understood before this kind of therapy becomes commonplace.

Microbes play an important role in gene therapy. Retroviruses are a family of viruses that produce an enzyme called reverse transcriptase. Retroviruses use reverse transcriptase to turn their RNA genome into DNA to add their genes into the host genome. Scientists have learned how to use retroviruses to reprogram human cells. The virus genome is modified to remove the disease-causing genes, which are replaced with genes that reprogram a patient’s cells. This treatment has been successfully used to treat leukemia by modifying the human immunodeficiency virus (HIV) to program the immune system to target the leukemia cells.

Retroviruses are not the only viruses used to deliver genes into a patient’s cells. However, only DNA delivered by a retrovirus enters the host genome. Several other types of viruses may be used to deliver genes into a patient’s cells and because the DNA delivered by these viruses does not enter the host genome, it does not produce a change that is passed down to daughter cells after mitosis. Adenoviruses are the most commonly used because they can efficiently deliver genes into a cell. Lentiviruses and the herpes simplex virus may also be used for gene therapies of this type, and which type of virus used depends on many factors. Different viruses are able to infect different cell types, for example herpes simplex virus targets neurons and the other types generally do not. The size of the genes that each type of virus will carry differs, herpes simplex virus can transfer larger genes, while lentiviruses cannot. However, adenoviruses and herpes simplex virus are more likely to trigger an inflammatory response than lentiviruses. When these viruses are used for gene therapy, the genes do not enter the host genome as is the case with retroviruses. A drawback of non-retroviral therapy is that it can only treat diseases with a temporary protein product. Currently adenoviruses are used to target cancer cells and introduce a gene causing apoptosis, the death of the cell. A herpes virus is used in a therapy to treat melanoma skin cancer.

Gene Therapy Using the Adenovirus

Adenovirus vectors can deliver genes into target cells where they are transcribed and translated into therapeutic products.

Single-Nucleotide Polymorphism (SNP) Applications

There are many individual bases in DNA that differ between organisms, and detection of these single-nucleotide polymorphisms (SNPs) is useful for an array of applications.

The most prevalent type of genetic variation is a single-nucleotide polymorphism. A single-nucleotide polymorphism (SNP) is a place in the genome where a single nucleotide is substituted for another—for example, a A for a G or a G for C. Mutations rates are low, yet genomes are very large, so mistakes during replication are relatively common, leading to a high prevalence of SNPs. For example, there are approximately 10 million SNPs in every human genome. In eukaryotes, SNPs occur in noncoding regions of DNA found between genes, so they tend to have little functional effect. They are, however, useful for a wide range of applications.

While SNPs do arise in individuals, the vast majority of SNPs in an organism's genome were inherited. By comparing the location of SNPs and the particular base at each location across different individuals in a population, it is possible to derive the relatedness or ancestry among individuals. Individuals that share more SNPs are more closely related. This process has been used to identify bacterial relatedness. Additionally, when SNPs occur in a gene-coding region, they can increase virulence factors or toxin potency. Researchers have been using SNPs to determine how virulence factors are impacted by genetics and to determine evolutionary development and biogeographic origins of pathogens. For example, a SNP in a strain of Mycobacterium tuberculosis, the causative agent of tuberculosis, is found in a membrane transporter and enhances its ability to move the antibiotic tetronasin across the plasma membrane and out of the bacterial cell.

Single-Nucleotide Polymorphism

A single-nucleotide polymorphism (SNP) is a place in the genome where a single nucleotide has been substituted for another. Individuals that share more SNPs are more closely related. For example, bacterial genomes that contain the same SNP are more closely related than those with different SNPs.
The correlation of SNPs with diseases or traits of interest is another application of SNP information. Plant breeders use SNPs as indicators of traits that they want to select, speeding the breeding process since the SNPs are identifiable before the plant has fully grown. When a particular SNP is associated with a trait of interest, the breeder can identify whether or not that SNP was inherited rather than waiting for the plants to grow to see if the trait was inherited. In many situations, searching for a SNP is faster and more cost effective than traditional methods of phenotyping. Similarly, many human diseases are associated with SNPs. A disease-related SNP might cause a disease if it is located within the coding region of a gene, such as with cystic fibrosis. More commonly researchers find associations between SNPs and a disease because they SNPs are physically located near the disease-gene, or genes, and are therefore inherited together. In these cases, studies documenting the SNPs and their effects on diseases and people's responses to treatment are valuable for furthering the understanding of diseases.