11.1Genetic Engineering

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The History and Development of Genetic Recombination

James Watson, an American researcher, and Francis Crick, a British researcher, discovered the double-helix structure of DNA in 1953. The two chains of DNA are paired with one another like the "positive" and "negative" of a photograph and play an important role in the replication of genetic information. The elucidation of this structure led to the development of molecular biology, which tries to unravel life phenomena based on information from the DNA.
In 1973, genetic recombination technology (recombinant DNA technology) emerged and became an important tool of molecular biology. Cohen and Boyer developed this technology, by which DNA is first cleaved at a specific position, and then the excised DNA fragment is incorporated and activated in another cell. This technology became possible through the discovery of "restriction enzymes," which act as "scissors" that cut the gene at a determined site. The discoverer of restriction enzymes later won the Nobel Prize. Although Cohen and Boyer did not win any Nobel Prize, Stanford University to which Cohen belonged received large revenues from the master patent on genetic recombination technology.
Genetic recombination technology has some fundamentally different features than biotechnology had so far.
First, by changing the genome (see Section 2 of Chapter 3) corresponding to the "blueprint" or "recipe" of a specific organism on the gene level, the properties of this organism can be changed on the blueprint level. In the case that gene recombination is performed on gametes or fertilized eggs, changes on the genetic level pass down to descendents. More specifically, it is possible to change the properties of some species.
The second point is that this technology made it possible to recombine genes among different species. As mentioned in Chapter 1, all organisms on earth, regardless of the differences in species, have DNA as the carrier of genetic information. Thus, human genes can be introduced into and activated in plants and microorganisms, and the reverse is also technically possible. This never happens by normal reproduction.

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The Asilomar Conference

When genetic recombinant technology emerged, scientists themselves voiced concerns regarding the risks of applying such techniques to living organisms. It was then decided that the specialists must put together specific guidelines for it first prior to performing gene recombination. They also published a "moratorium" in the scientific journal - Science - to suspend the use of this technology until the guidelines were developed.
In response to this appeal, in 1975 in Asimolar, United States, a conference was held at which biologists from around the globe came together to discuss gene recombinant technology. The Asilomar Conference is regarded as an epoch-making event in the field of the application and regulation of science and technology. This is because until then science and technology had meant nothing but development and —application of technologies, and scientists—the central players of development—had never attempted to self-regulate the application of scientific technologies.
At the Asilomar Conference, the scientists came to an agreement that as long as biological and physical containments are appropriately executed, almost all recombinant DNA experiments should continue, but high-risk experiments must be rescheduled to a later date. Biological containment refers to the methods that ensure safety when recombinant DNA cells and DNA carrying vectors*1 are combined, while physical containment refers to a laboratory so structured that recombined organisms cannot get outside.
Based on this agreement, the guidelines for DNA experiments were established in each country, and experiments were conducted within these guidelines.
In 2003, the Cartagena Protocol on biodiversity came into effect, and as the domestic law corresponding hereto, the Law for the Securement of Biodiversity by Regulating the Use of Recombinant Organisms etc. was enacted. This law, in place of the experimental guidelines on recombinant DNA, regulates the handling of recombinant organisms..

*1 In recombinant DNA experiments, a "carrier" such as virus DNA is used for introducing DNA into cells.

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Production of Useful Substances

Genetic technology has developed in various directions. Some typical examples include: production of useful materials such as pharmaceutical products by genetic recombination, creation of genetically modified crops and animals, and functional analysis of the human genome and its medical application.
For the production of useful materials by recombination techniques, Escherichia coli and yeast have been used. Besides DNA, which carries the main genetic information, E. coli bacteria and yeast contain a small circular piece of DNA called a "plasmid." Genes of other organisms can be incorporated into plasmids, which are then introduced into E. coli bacteria, and the incorporated genes can then be activated. For example, by multiplying E. coli bacteria containing an incorporated human insulin gene on a large scale, a mass production of insulin can be made. Insulin, which is used for the treatment of diabetes, was previously extracted from porcine and bovine pancreas for use.
Ever since human insulin was successfully produced by genetic recombination in 1980, genetic recombination has also been applied to the production of useful materials such as growth hormones and interferon.
Nevertheless, even though a human gene is incorporated, this does not necessarily mean that exactly the same protein can be produced as the one generated in the human body. It's because the conformation may be different, or other molecules inside of the human body may be missing. Also, the elimination of E. coli-derived impurities requires a cautious and laborious process. In an effort to produce highly safe materials efficiently, genetic recombination using insect and animal cells has also been demonstrated.

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Genetically Modified Organisms

Genetically modified crops are mainly produced with a bacterium called Agrobacterium. Agrobacteria contain plasmids and have the property to insert their plasmids into the genes of plants, with which to live in symbiosis. By incorporating foreign genes into such plasmids, these genes can be activated in plants.
Some crops such as soybeans, cotton, rapeseed, and corn are available in the market as "pest-resistant" crops (Fig. 11-1) and "herbicide-tolerant" crops. Pest-resistant crops are developed by introducing the gene of a protein called Bt toxicity containing insecticidal activity in the crops, whereas herbicide-tolerant crops are developed by introducing a gene resistant to certain herbicides in the crops. Currently, these genetically modified crops are also available in the market as processed goods.

Fig. 11-1. Schematic Diagram of the Creation of Genetic Recombinant Crops (pest-resistant corn)

How safe are such recombinant crops? In Japan, before food products, animal feedstuff, and additives become available in the market, the governmental Food Safety Commission demands submission of scientific data from the developer and assesses the safety of these products. For safety evaluation, recombinant crops are assessed in comparison with the usual crops without the introduced gene. Presently, besides creating a product of an introduced gene, recombinant crops can therefore be considered scientifically safe only if their properties are almost identical with those of the original crop.
Furthermore, the possibility that the proteins synthesized by recombinant crops might have biological effects has also been investigated. For instance, it has been scientifically demonstrated that in case of pest-resistant crops, the protein produced by the Bt toxic gene is only toxic to insects and does not affect mammalians. Currently, it is believed that in humans, the Bt toxic protein has almost no problems regarding digestion, or the development of allergies.
Even after such a safety review, many people still have a negative image of genetic manipulation itself, and fear that it may affect their health and the environment in the long run. Admittedly, the history of recombinant crops is short, and thus, the fear about such crops cannot be considered immaterial.
On the other hand, such technology may help decrease the distribution of herbicides and agricultural chemicals. It has also been pointed out that food shortage could be addressed by the development of "drought-resistant" crops that can grow in the desert and "cold-resistant" crops that are resistant to a cold environment. It is vital to judge the validity of recombinant crops by balancing their potential benefits and risks.


Genetically Modified Foods in Japan

Half of all genetically modified (GM) soybeans, corn, cotton, and rapeseeds, which have herbicide-tolerant or pest-resistant qualities, are cultivated in the United States. Such cultivations are on the increase in other countries such as in Argentina, Brazil, India, and China. Ninety percent of soybeans produced in the United States are GM soybeans. Today, these types of crops do not directly contribute to food provision in the developing countries, but help yield profits for seed-developing companies and farm producers through stabilized crop productions and reduced workforce and costs.
Currently, Japan's food self-sufficiency is 40%, and except for rice, almost all major grains need to be imported. As a result, Japan depends on GM crops. In the country, many GM crops are recognized as safe food products, but GM crops are not yet commercially cultivated in the country because it is believed that consumers find it hard to understand all the advantages of GM food products, and also, they feel quite challenged to try them.
In 2001, the Law of Japan Agricultural Standards (JAS) was revised; a uniform labeling system of GM food products sold in Japan was made compulsory. However, food products to which 5% or less of GM varieties are admixed require no labeling. Labeling indicates the purity of commercial products, but not the degree of safety. Furthermore, commercial products such as dietary oils, which cannot be tested for whether or not the raw material is a GM variety, need no labeling. For these reasons, GM food products widely circulate in Japan, and people are consuming them daily without being aware of it.
For a long period of time, the focus of the debate on GM food products has been on their safety. But thus far, unexpected toxins have neither been expressed nor admixed. Methods that can examine allergens have also been established. Yet, 100% safety of GM food products cannot be guaranteed. By the same token, not recombinant, conventional food products are not 100% safe either. The safety evaluation of GM food requires not only greater efforts to gain better understanding of how GM food products can be scientifically tested, but also appropriate communication for risk management in order to make optimal judgments under uncertain, unpredictable conditions.
In 2004, when the domestic law relating to the biodiversity treaty was enforced, the center of debate began to switch to the contamination of indigenous varieties with GM food products, and the gene infiltration of wild and related species. From the aspect of quality control and assurance, and also the aspect of consumers' right to choose, what the general public desires is to find methods that can segregate measures for GM and other crops used in food production and circulation.
Besides conventional GM food products, the technologies that enable consumers to directly avail their benefits are being developed nowadays. For instance, rice containing large amounts of a precursor of vitamin A has been developed overseas. In Japan, rice that alleviates pollen allergy has been developed as a non-food product; however, it needs to go through an approval process to be completely recognized as a pharmaceutical agent.

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Genetically Modified Animals

Recombinant technology has also made introduction of genes into animals possible. Theoretically, this also allows for improvement through breeding; however, thus far, no genetic recombinant meat or fish been introduced in the market. Rather, genetic recombinant mice are widely used as test animals (Fig. 11-2).
There are various kinds of recombinant mice. A mouse raised by incorporating foreign genes into fertilized eggs is called a transgenic mouse. Generally, this is done by a method by which one injects the target DNA into a fertilized egg using a fine glass pipe while observing the procedure through a microscope. However, this method cannot enable genes to be introduced at a targeted position on the chromosome. Compared to ordinary organisms, mice genes often move excessively.
The method that can alter target genes is called gene targeting. This method is able to destroy genes and produce a "knockout mouse." In order to create such a mouse, embryonic stem cells (ES cells) generated from fertilized eggs can be used (ES cells are discussed later. See also Chapter 5). The techniques by which target genes can replace other genes have also been developed.

Fig. 11-2. Creation of a Genetic Recombinant Mouse

There are 2 types of genetic recombinant mice: a "transgenic mouse" and a "knockout mouse." In order to create a knockout mouse, first, ES cells, in which the target gene has been destroyed, are injected into the embryonic cells of a separately prepared mouse (during the phase when fertilized eggs have grown for several days). These cells, after being nourished in the uterus of a surrogate mother, eventually grow into a chimeric mouse, and this mouse is a product of combined derivative cells of ES and fertilized eggs. In the body of the chimeric mouse, normal cells and cells, in which one target gene of homologous chromosomes was destroyed, are mingled. When reproductive cells are crossed with an ES cell-derived chimeric mouse and a normal mouse (wild type), a mouse (+/-) with cells, in which one allelic target gene has been destroyed, is produced. When this mouse is crossed, a knockout mouse (-/-), in which both allelic target genes are destroyed at a fixed percentage, is produced.

Both kinds of recombinant mice are used in research to discover how specific genes work in individual organisms. By incorporating or destroying genes related to certain diseases, a "disease model" mouse can also be replicated.
However, as with microorganisms and plants, since such methods can create animals that do not exist in nature, to begin with, it is imperative that these animals should be prevented from being released into the environment. Therefore, when recombinant animals are being used, similar to recombinant crops, their risks and benefits must be weighed against each other.

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The Bright and Dark Sides of Genetic Testing

At the beginning of the 1980s, search for human genes became more active. In particular, attention was paid to the genes that were the cause of diseases. Human genetic disorders are caused by mutations and chromosomal abnormalities inherited by the children from their parents. In order to trace down mutations of the genes that cause genetic disorders, family analysis has been carried out. In recent years, besides study on genetic disorders, continued vigorous analyses of the genes of lifestyle-related diseases such as cancer, diabetes, and hypertension have been underway.
The genetic analysis of diseases has made the development of a technique called "genetic diagnosis" possible.
Genetic diagnosis, which has thus far been made available, is mainly intended for hereditary disorders. There are various types of diagnosis, among which particularly strong impacts they have on society are: preclinical diagnosis, prenatal diagnosis, and preimplantation diagnosis (see Column below).
Huntington disease is often taken as an example of preclinical diagnosis. It is an autosomal dominant genetic disorder (see Section 3 of Chapter 3) that mainly manifests itself in middle age and causes severe neurological symptoms. At present, there is no treatment for this disease. The gene abnormality that causes this disease was discovered in 1993, and genetic diagnosis of this disorder has since become possible. However, a positive diagnostic outcome means the pronouncement of an incurable disease whose manifestation is almost certain in the future. Therefore, the diagnosis must be done with caution.
Prenatal diagnosis is a technique by which the baby is tested before birth, whereas preimplantation diagnosis is a technique by which eggs that have been fertilized in vitro are genetically diagnosed. Both techniques are mainly conducted for the purpose of "avoiding the birth of a child with severe genetic disorder,"but there is also some criticism that" fetal diagnosis may encourage abortion" and "either of these diagnoses leads to the discrimination of disabled persons" (see also Chapter 5).
As mentioned above, genetic diagnosis has mainly been conducted for "single gene disorders," which literally means a disorder is dominated by a single gene. Today, however, there is also research that aims at the genetic diagnosis of lifestyle diseases, wherein the manifestation of a disease is influenced by multiple genes and the environment. The possibility to diagnose the risk of lifestyle diseases with SNPs (see Section 4 of Chapter 3 [3.4.1]), which are associated with individual genetic differences, is particularly attracting attention.


Prenatal Diagnosis and Preimplantation Diagnosis

Prenatal diagnosis is a procedure by which amniocentesis and chorionic villus sampling is performed, and the fetus-derived cells contained therein are examined for chromosomal and genetic abnormalities. This procedure is mainly demonstrated for the purpose of avoiding the birth of a child with a severe disease, but there is also the risk of miscarriage.
In preimplantation diagnosis, some cells of the embryo that have been produced by in vitro fertilization are collected, and then chromosomes and genes are examined. While being employed to avoid the birth of a child with a severe disease, this procedure can also applied to a couple with abortus habitualis caused by chromosomal abnormalities. Furthermore, there are some cases in which this method is used to help a child who requires bone marrow transplantation by selecting a fertilized egg with an identical HLA type (human leucocyte antigen type) when the mother becomes pregnant and gives birth to a baby.
For either of these procedures, there are no regulations at a national level, and guidelines are determined at the level of academic societies.

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Gene Therapy

Genetic recombinant technology has also made human gene therapy possible. The original idea of gene therapy (see Fig. 3-9, Chapter 3) was to "replace defective genes that cause disease with normal genes." At present, the replacement of genes is technically difficult, and medical strategies, i.e., adding normal genes or genes that fight the disease, are being applied.
Upon implementation of gene therapy, besides its safety, the pros and cons of the act of recombining human genes itself have also been the focus of debate. There are strong critical opinions that human nature will be changed by genetic recombination, and gene therapy manipulating the genes of fertilized eggs or gametes should be forbidden worldwide so that artificially added genetic changes cannot be inherited by the descendants.
In case of gene therapy of somatic cells, genetic changes are limited to the present generation. In Japan, this procedure is only permitted for treatments of severe illnesses and diseases that markedly influence a person's quality of life. Today, however, there are only few successful cases.


Ethical Issues in Genetic Recombination

When genetic recombinant technology was introduced and then discussed at conferences such as the Asilomar Conference and the like, scientists mainly paid attention to the problem of "safety" of this technology. The general public was also interested in the ethical issues concerning the modification of genes, which is the blueprint of life.
Even though there is no resistance regarding the genetic modification of microorganisms, there may still be people who feel resistance to genetic modification of higher organisms. For example, by introducing a gene related to bioluminescence into fish, "luminous fish" can be produced. Moreover, a luminous mouse, into which luminescence genes were introduced, has already come into existence. Basically, all these have been done for research purposes, and it is possible to grow luminescent aquarium fish and animals for ornamental purpose. The question is: to what extent should the application of such genetic engineering be permitted?
In reality, gene therapy can be equated with genetic recombination of humans. The idea of giving birth to a child with genes according to one's wishes is called a "designer baby," and it is under debate. Again, the question is: to what extent should the genetic recombination of humans be allowed?

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The Human Genome Project

In the second half of the 1980s, the unfamiliar term "human genome" began to appear in the general media. As mentioned in Chapter 3, the word genome refers to all the genetic information possessed by an organism (Fig. 11-3). An attempt to decode the entire genetic information of man, i.e., the possibility of a "human genome project," began to be discussed in the 1980s.
The first stage of the human genome project was to determine all base sequences of DNA constituting the human genome. In contrast to the methods used thus far by which scientists had manually decoded portions of the genome that they were interested in, this project tried to decode the length and breadth of the entire human genome, thus obtaining the complete human blueprint. Until that point, in the field of biology, scientists tended only to concentrate on the genes in which they were interested. In this sense, this project was fundamentally different from the conventional research in the past, and generated a lot of debate concerning its methodology and the right to access data.
At the end of the 1980s, the United States started the human genome project as a national agenda. With this as a starter, Japan, Europe, Canada and others participated in this project, and the decoding of the human genome proceeded as a publicly acknowledged, international joint project. In order to avoid overlapping analysis, separate research groups were formed with assignments allocated to each. It was also agreed that the decoded data would turn into a public database.
The American molecular biologist Craig Venter caused a stir to this international joint project. He set up a venture company to conduct genome analysis and tried to get patents and sell data, while competing with the official genome decoding team. In 2000, competition between the "administration" and the "private sector" ended politically, with public announcement of their joint research on the "Draft sequence of the human genome (digest version)." In 2003, the international joint team of the project announced completion of the decoding of the human genome.
The decoding of the human genome confirmed that the entire base sequence of DNA constituting the human genome is about 3 billion and the number of genes is about 25,000–less than our expectations.

Fig. 11-3. Schematic Diagram of the Human Genome



When the human genome project entered its final stage, the framework for a "biobank" emerged in each country. A biobank collects information on the blood and diagnosis of a multitude of people, and examines how an individual's genetic make-up and environmental factors are linked to the development of specific disorders and the action and adverse effects of specific pharmaceutical agents.
In Iceland, where the idea of a biobank was pioneered, laws were developed for compiling the diagnostic records and DNA information of the whole nation into a database. At one point, the country decided to give the exclusive right of utilization of this data bank to the private company deCODE genetics Inc. for a certain period of time in exchange for giving them the charge of maintenance of the database. However, this approach has been strongly criticized inside and outside of Iceland. This case asks us the questions: who has the right to handle individual genetic information, and what steps need to be taken as far as their use is concerned.
In Japan, under the Realization of Order-Made Medicine Project by the Ministry of Education, Culture, Sports, Science and Technology, the set-up of a biobank began in 2003 as a 5-year project. The project goal was to collect DNA and blood serum samples of 300,000 cases with focus on patients with lifestyle diseases. With the biobank in full operation, a department in charge of ethical, legal, and social issues was also set up so that they can deal with issues such as appropriate informed consent, protection of personal information, and access right to the bank.
With samples from the biobanks, the search for a method that can discover genes pertaining to human disorders and the responsiveness to medical agents was conducted, and this operation was called the International HapMap Project.
Today, various kinds of biobanks exist, which collect biological samples and material for different purposes, such as cell banks, test animal banks, and sperm banks for reproductive medicine.


Technique for Amplifying Minute Amounts of DNA: PCR

For analyzing, cleaving, DNA, certain amounts of DNA are required. However, the amount of DNA that is contained in one cell is extremely small, and it is difficult to handle DNA if only small amounts of sample are available.
In the 1980s, Richard Morris developed a polymerase chain reaction (PCR) technique that solved this problem. By the PCR technique, only a specific DNA fragment can be amplified exponentially (Column Fig. 11-1). The device used for this technique is now indispensable in laboratories that handle DNA. Additionally, it is also used for DNA analysis with minute amounts of DNA (see Column at the bottom).
At the same time, due to its high sensitivity, there is a risk involved that unintended DNA fragments can be amplified, which calls for the necessity of careful handling of samples.

Column Figure 11-1. Fundamentals of PCR

Prepare the following types of DNA: a double-stranded DNA with a piece of DNA targeted for amplification and DNA polymerase, a single-stranded DNA fragment (primer) corresponding to both sides of the piece of DNA targeted for amplification. Keep them in a solution. When the temperature of the solution rises up to about 90 degrees Celsius, the double-stranded DNA unfolds (1). When the temperature drops to 50 degrees, the primer sticks to the unfolded DNA (2). When the temperature rises to 70 degrees, DNA is synthesized with the primer as a starting point, using the single-stranded DNA as a template (3). By repeating this cycle, the target piece of DNA can be amplified.

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Ethical Issues Involving the Human Genome and Genetic Analysis

The Asilomar Conference was already epoch-making in that scientists themselves considered regulations for genetic engineering, but even more so when unprecedented proposals on the human genome project were made in the United States. As mentioned in Chapter 10, the proposal was to regard research on the ethical, legal, and social issues (ELSI) arising from the decoding of the human genome as part of the human genome research, and to allocate 3–5% of the budget for the human genome project to such areas of study.
In the United States, this proposal came into effect and helped generate a multitude of research projects. Moreover, this effort contributed to setting up of bioethics centers at universities all across the country.
In Japan, the launching of the Millennium Genome Project by the government in 1999 allowed its citizens to get an up-close look at ethical issues concerning the human genome and gene analysis. Ethical principles of human gene analysis were first drafted for the Millennium Genome Project, and in 2001, the three ministries—the Ministry of Health, Labor and Welfare; the Ministry of Education; and the Ministry of Economy, Trade and Industry—together formulated Ethical Guidelines Regarding Research on the Human Genome and Gene Analysis on a national level.
These guidelines stipulate that prior to conducting human genome analysis, it is compulsory to obtain informed consent from the test subject, and that the research project and letter of consent are to be examined by an institutional ethics committee (see Chapter 10).


DNA Profiling

Among humans, 99.9% of the human genome is common, and individual differences occur in the remaining 0.1%. This difference of 0.1% brings about distinctive individual characteristics.
By analyzing individual differences in DNA, it is also possible to identify individuals and to determine parentage. This technique is called "DNA analysis."
For example, for criminal investigation, one takes the DNA of blood left at a crime scene, compares it to the DNA of a crime suspect, and uses the results to judge whether they belong to the same person (Column Fig. 11-2).
Any person inherits the DNA of his or her parents, and thus, the characteristics of a child's DNA absent in the mother's DNA should be present in the father's DNA. This principle is now put in use: DNA testing for "paternity case" is used at trials in court to identify a child's father.

Column Figure 11-2. Schematic Diagram of DNA Analysis

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