Foundations of Genetic Engineering
Microbial genetics, which emerged in the mid-1940s, was based upon the principles of heredity that were originally discovered by Gregor Mendel
in the middle of the nineteenth century and the resulting elucidation of the principles of inheritance and genetic mapping during the first forty years of the twentieth century. Between the mid-1940s and the early 1950s, the role of DNA as genetic material became firmly established, and great advances occurred in understanding the mechanisms of gene transfer between bacteria. The discovery of the structure of DNA by James Watson and Francis Crick (aided by the x-ray photography of Rosalind Franklin) in 1953 provided the stimulus for the development of genetics at the molecular level, and, for the next few years, a period of intense activity and excitement evolved as the main features of the gene and its expression were determined. This work culminated with the establishment of the complete genetic code in 1966, which set the stage for later advancements in genetic engineering.
Initially, the term “genetic engineering” included any of a wide range of techniques for the manipulation or artificial modification of organisms through the processes of heredity and reproduction, including artificial selection, control of sex type through sperm selection, extrauterine development of an embryo, and development of whole organisms from cultured cells. However, during the early 1970s, the term came to be used to denote the narrower field of molecular genetics, involving the manipulation, modification, synthesis, and artificial replication of DNA in order to modify the characteristics of an individual organism or a population of organisms.
The Development of Genetic Engineering
Molecular genetics originated during the late 1960s and early 1970s in experiments with bacteria, viruses, and free-floating rings of DNA found in bacteria, known as plasmids. In 1967, the enzyme DNA ligase was isolated. This enzyme can join two strands of DNA together, acting like a molecular glue. It is the prerequisite for the construction of recombinant DNA molecules, which are DNA molecules that are made up of sequences not normally joined together in nature.
The next major step in the development of genetic engineering came in 1970, when researchers discovered that bacteria make special enzymes called restriction endonucleases, more commonly known as restriction enzymes. Restriction enzymes
recognize particular sequences of nucleotides arranged in a specific order and cut the DNA only at those specific sites, like a pair of molecular scissors. Whenever a particular restriction enzyme or set of restriction enzymes is used on DNA from the same source, the DNA is cut into the same number of pieces of the same length and composition. With a molecular tool kit that included isolated enzymes of molecular glue (ligase) and molecular scissors (restriction enzymes), it became possible to remove a piece of DNA from one organism’s chromosome and insert it into another organism’s chromosome in order to produce new combinations of genes (recombinant DNA) that may not exist in nature. For example, a bacterial gene could be inserted into a plant, or a human gene could be inserted into a bacterium.
The first recombinant DNA molecules were generated by Paul Berg at Stanford University in 1971, and the methodology was extended in 1973 by Stanley Cohen and Herbert Boyer, who joined DNA fragments to Escherichia coli
(E. coli) plasmids. These recombinant molecules could replicate when introduced into E. coli cells, and a colony of identical cells, or clones, could be grown on agar plates. This development marked the beginning of the technology that has come to be known as gene cloning, and the discoveries of 1972 and 1973 triggered what became known as “the new genetics.” In 1977 two methods for sequencing DNA were published by Allan Maxam and Walter Gilbert, and by Frederick Sanger and his associates, allowing for the sequencing of coded proteins. Berg, Gilbert, and Sanger were awarded the 1980 Nobel Prize in Chemistry. These technologies, coupled with the power of computer processing and database analyses, set the stage for genome sequencing. The use of the new technology spread very quickly, and a sense of urgency and excitement prevailed. However, because of rising concerns about the morality of manipulating the genetic material of living organisms, as well as the fear that potentially harmful organisms might accidentally be produced, US biologists called for a moratorium on recombinant DNA experiments in 1974. That same year the secretary of the Department of Health, Education and Welfare, now known as the Department of Health and Human Services, created the Recombinant DNA Advisory Committee to oversee rDNA research. In 1976 the National Institutes of Health (NIH) issued the Guidelines for Research Involving Recombinant DNA Molecules to control laboratory procedures for gene manipulation. They were revised in 1978, 2002m and 2013.
In 1977, the pioneer genetic engineering company Genentech produced the human brain hormone somatostatin, then in 1978 produced human insulin in E. coli by the plasmid method of recombinant DNA. Human insulin was the first genetically engineered product to be approved for human use. By 1979, small quantities of human somatostatin, insulin, and interferon were being produced from bacteria by using recombinant DNA methods. Because such research was proven to be safe, the NIH gradually relaxed the guidelines on gene splicing between 1978 and 1982. The 1978 Nobel Prize in Physiology or Medicine was shared by Hamilton O. Smith, the discoverer of restriction enzymes, and Daniel Nathans and Werner Arber, the first people to use these enzymes to analyze the genetic material of a virus.
By the early 1980s, genetic engineering techniques could be used to produce some biomolecules on a large scale. In December 1980, the first genetically engineered product was used in medical practice when a diabetic patient was injected with human insulin generated in bacteria; in 1982 the Food and Drug Administration (FDA) approved the general use of insulin produced from bacteria by recombinant DNA procedures for the treatment of people with diabetes. During the same time period, genetically engineered interferon
was tested against more than ten different cancers. Methods for adding genes to higher organisms were also developed in the early 1980s; genetic researchers succeeded in inserting a human growth hormone gene into mice, which resulted in the mice growing to twice their normal size. By 1982 geneticists had proven that genes can be transferred between plant species to improve nutritional quality, growth, and resistance to disease.
In 1985, experimental guidelines were approved by the NIH for treating hereditary defects in humans by using transplanted genes. The more efficient polymerase chain reaction (PCR) cloning procedure for genes, which produces two double helixes in vitro that are identical in composition to the original DNA sample, was also developed. The following year, the first patent for a plant produced by genetic engineering, a variety of corn with increased nutritional value, was granted by the US Patent and Trademark Office. In 1987, a committee of the National Academy of Sciences concluded that no serious environmental hazards were posed by transferring genes between species of organisms, and this action was followed in 1988 by the US Patent and Trademark Office issuing its first patent for a genetically engineered higher animal, a mouse that was developed for use in cancer research.
Human Genome Project
The Human Genome Project
was proposed in 1985 by Charles DeLisi, director of the Office of Health and Environmental Research at the Department of Energy (DOE), to better understand potential changes to human DNA in the aftermath of the atomic bombs dropped by the United States on Nagasaki and Hiroshima, Japan, to end World War II. Sequencing began in 1990 in an international effort to map all of the genes and 3.1 billion base pairs on the human set of twenty-three pairs of chromosomes. Since 1995, thousands of organisms have been sequenced, providing valuable data for comparative studies of genetic disorders. In 2007, Sir Martin John Evans of Cardiff University was awarded the Nobel Prize for creating chimeric, or transgenic, mice genetically engineered to lack a targeted “knockout” gene, a model particularly useful for understanding the genetics of cancer and psychiatric disorders. In April 2009, a research team led by Byeong-Chun Lee of Seoul National University in South Korea announced the cloning of the world’s first litter of transgenic puppies. Ruppy the ruby puppy and her littermates express a red fluorescent gene produced by sea anemones, allowing them to glow in the dark. The mapping of the dog genome sequence provides researchers new material for unraveling the mechanics of human disease. Data bioinformatics systems continue to provide complex arrays and algorithms for mapping genetic characteristics. In 2008, President George W. Bush signed H.R. 493, the Genetic Information Nondiscrimination Act (GINA), prohibiting discrimination on the basis of genetic testing.
Impact and Applications
The application of genetic engineering to gene therapy
(the science of replacing defective genes with sound genes to prevent disease) is still in the formative stages of clinical trial. Early trials introducing genes straight into human cells often failed, intensifying a wary public distrust of gene therapies. In 1990, genetically engineered cells were infused into a four-year-old girl to treat her adenosine deaminase (ADA) deficiency, an inherited, life-threatening immune deficiency called severe combined immunodeficiency disorder (SCID). In 1991, gene therapy was used to treat skin cancer in two patients. In 1992, small plants were genetically engineered to produce small amounts of a biodegradable plastic, and other plants were manufactured to produce antibodies for use in medicines.
By the end of 1995, mutant genes responsible for common diseases, including forms of schizophrenia, Alzheimer’s disease, breast cancer,
and prostate cancer, were mapped, and experimental treatments were developed for either replacing the defective genes with working copies or adding genes that allow the cells to fight the disease. During the sequencing of the human genome, genes were identified for cystic fibrosis, neurofibromatosis, Huntington’s disease, and breast cancer. In 1997, a lamb named Dolly was cloned from the DNA of an adult sheep’s mammary gland cell; it was the first time scientists successfully cloned a fully developed mammal. By the end of 1997, approximately fifty genetically engineered products were being sold commercially, including human insulin, human growth hormone, alpha interferon, hepatitis B vaccine, and tissue plasminogen activators for treating heart attacks. In 1998, strong emphasis was placed on research involving gene therapy solutions for specific defects that cause cancer (including the discovery of oncogenes), as well as on a genetically engineered hormone that can help people with damaged hearts grow their own bypass vessels to carry blood around blockages. In 2003, genes were successfully inserted into the brain, a potential therapy for Parkinson disease. In 2007, the world’s first gene therapy for retinal disease was announced. Since 2010, studies have taken place in which gene therapy has been used to treat patients with HIV, leukemia, and multiple myeloma.
In 1999, Jesse Gelsinger, a healthy eighteen-year-old participating in a gene therapy clinical trial at the University of Pennsylvania, died unexpectedly, casting doubt on the safety of some types of gene therapy. In another set of clinical trials in France in 2002, involving the treatment of children with SCID, two of the children developed leukemia, raising doubts about the safety of yet another gene therapy protocol. In 2003, the FDA regulated against the use of retroviral vectors in stem cells. Continuing research using nanotechnology, viral vectors, lymphocytes, RNA interference, transcriptional profiling, protein analysis, and epigenetic response to the environment continues to strengthen the prediction and treatment of human disease.
Key terms
chimera
:
a transgenic organism
clone
:
a group of genetically identical cells
DNA sequencing
:
the process of determining the exact order of the 3 billion base pairs constituting the human genome
plasmids
:
small rings of DNA found naturally in bacteria and some other organisms
polymerase chain reaction
:
also called DNA amplification, it is a laboratory process first developed in 1983 by Nobel laureate Kari Mullis to replicate DNA fragments in batches large enough for analysis and manipulation
protein-ligand complex
:
the structural description of how proteins interact with other proteins, RNA, DNA, and other small molecules; understanding the role of small molecules and their effects on protein docking are essential to drug and gene therapies
recombinant DNA
:
a DNA molecule made up of two or more sequences derived from different sources
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