Good-bye Normal Gene

Genetic engineering refers to "cutting and pasting" of genetic material.

In gene transfer, a string of genes from a microorganism, animal, or plant is placed in the chromosome of an animal or plant, causing it to display the special characteristics of that gene. This differs from traditional cross-breeding in that gene transfer can completely transcend species barriers. Genes can be interchanged freely among microorganisms, animals, and plants.

Animal cloning, on the other hand, means that a biological entity is reproduced not through sexual reproduction, but through nuclear transfer techniques.

Asexual reproduction is not uncommon in the plant kingdom, but in the animal kingdom it is the "creature" of genetic engineering technology. The sheep Dolly was the first successfully cloned animal in the world.

At present, there are not many cloned animals from somatic cells. Besides Dolly, other successful examples have involved cattle (in Japan) and mice (in Hawaii). As for Taiwan, things are still in the experimental stage. On the other hand, gene transfer is spreading like wildfire around the world.

（above） DNA micro-injection in a mouse embryo. Mice are the most frequently chosen animals for gene transfer research. （courtesy of PRIT）

The path of the future?

A report in the June issue of Nature magazine stated that a research team at Cambridge University isolated a gene from mouse-ear cress (Arabidopsis thaliana) that encourages cell division. After the transfer of this gene into tobacco leaf, this gene produces protein which, after combining with certain natural materials, causes the cells at the tip of the rhizome to divide rapidly. This genetically engineered tobacco grows twice as fast as ordinary tobacco.

In recent days, newspapers in Taiwan have reported that some businessmen are importing "super poplar trees" to address the landslide and soil problems facing central Taiwan in the wake of the September 21 earthquake. It is said that these poplar trees, a product of gene transfer, grow from 3 to 5 centimeters per day on average. In a year they can grow 6 to 7 meters, and become towering giants within only two or three years.

Fast-growing tobacco, three-year giant poplars, and the like are all products of genetic engineering. In fact the types of agricultural crops treated by gene transfer far exceed tobacco and poplars. They include beans, corn, rape, cotton, tomatoes, rice, potatoes, fruit trees, and more.

The purpose of gene transfer varies. For some of these (tomatoes), the purpose is to prevent rotting; in others (beans, corn, cotton) it is to strengthen resistance to the effects of herbicides; and in others (corn, potatoes) it is to increase resistance to insects.

In the past, agriculture typically involved using a large amount of pesticides, herbicides, and fertilizers. These leave large amounts of chemical residues in the soil, which are harmful to humans and the environment. Some say that the use of transgenic crops can solve this problem. Others say that transgenic crops will be able to resolve a looming food shortage that may face mankind in the next century.

（below） A newly fertilized pig embryo has DNA injected into the sperm nucleus using the micro-injection method. This is "genetic engineering." （courtesy of PRIT）

Insect-resistant and nutritious

Chen Ching-san, a researcher in the Institute of Botany at the Academia Sinica, is currently working on gene transfer research for paddy rice. He hopes on the one hand to improve the quality of the rice, and increase its protein content. On the other, he wants to make the rice more resistant to disease and insects, so that insecticide use can be reduced and production volume increased.

He has used the bacillus that was originally a parasite in the plant as a vector to transfer the bug-resistant gene of the green bean into paddy rice. Tests show that the gene has indeed been transferred into the rice, but has not taken the next step to produce proteins. Chen Ching-san infers that the reason for this may be that the relationship between the genetic materials of beans and paddy rice is too remote.

Besides crops, there have been remarkable successes in gene transfer for flowers.

Chen Wen-hui, deputy-director of the Agri-Business Department at Taiwan Sugar Corporation, which is famous for its orchid exports, relates that Taiwan Sugar has successfully transplanted virus- and bacterial-soft-rot-resistant genes in the butterfly orchid.

Chen points out that the butterfly orchid has long been susceptible to bacterial soft rot. Inter-breeding of various types has never been able to resolve the problem. Presently they are transferring a bacterial-soft-rot-resistant gene that can be found in tobacco, in hopes this will solve the problem. So far, the virus-resistant gene has already been introduced into the butterfly orchid, while the soft-rot fighting gene is still in a trial stage.

In addition, Taiwan Sugar's Agricultural Research Institute has more than a dozen researchers working exclusively on flowers. They are currently studying the DNA molecular marker for flower colors. They hope that as early as the budding stage they will be able to know what the future color of the flower will be.

Besides gene transfer that enhances resistance to bacterial and insect infections, or alters the character for agricultural applications and flowers, another use is to produce medicinal molecules.

"No living thing can produce the things that we can make by gene transfer," says Shaw Jei-fu, director of the Institute of Botany at the Academia Sinica. Moreover, gene transfer promises to make these things faster and cheaper. For example, plant photosynthesis requires only the simplest ingredients-sunlight and water is enough. If, for example, blood coagulants or high value proteins can be transferred into plants, this would be a low-cost high-value-added form of production.

Currently, in Japan, carotene has been successfully transplanted into rice, and elsewhere vaccines have been transplanted into bananas. With these, you can help your eyes buy eating rice and immunize yourself against disease by eating bananas.

Genetic engineering aims for useful results, such as planting insect-resistant genes in crops.

Cross-species organ transplants

Currently, work in transgenic animals is tending toward three directions: improving livestock, organ transplants, and pharmaceutical research and development.

Among the goals in the first category are reducing the amount of fat in meat, raising the quality and production of dairy products, and increasing the growth rate of livestock.

Simon J.T. Mao, department head of applied biological sciences at the PRIT, says that currently the PRIT is using pigs to produce pharmaceutical molecules and organs similar to those of humans.

Mao's colleague Tu Ching-fu has been studying cross-species organ transplants (xenotransplantation) for many years. He notes that the early organ transplant research program using non-human primates was quite successful. But because such primates are protected, they had to settle for the next best thing. Now, pigs, which are anatomically and physiologically similar to humans, are the focus of research.

"The biggest problem with transplanting pig organs in human recipients is rejection," said Tu. With the exception of Old World primates (humans, apes, and Old World monkeys), the blood vessels in all other mammals have a type of galactose on the outside of the cell membrane. The antigens of the galactose induce a "hyperacute rejection response" which must somehow be controlled.

But even if this hurdle is cleared, there are still potential problems of acute or chronic rejection, caused by vascular coagulation and the cell-mediated immune system. The PRIT is currently trying to create a transgenic pig which could overcome acute and chronic rejection. Their work fits in well with studies currently being conducted in the United States and Europe on preventing hyperacute rejection.

Tu Ching-fu points out that there is evidence showing that the transgenic pigs are more suitable for organ transplants than the non-transgenic pigs. They plan to cross-breed the HLA II pig raised at the PRIT with the hDAF transgenic pig being raised in Europe and the States. This would be a major step forward for cross-species organ transplantation.

Resistance to disease or insects, or alterations to the basic nature of a living thing, can all be accomplished by gene transfer. The photo shows transgenic tobacco, which grows twice as fast as the original.

Animal factory

Using genetic engineering to improve the economic value of livestock is a direction being pursued by the animal product industry worldwide.

The Council of Agriculture and the National Science Council have commissioned the PRIT and National Taiwan University to do a joint project to manufacture transgenic pigs that will increase the amount of protein in the milk that sows feed to their young. There has already been some preliminary success.

Wu Shinn-chih of the PRIT's applied biology department, who is involved with this project, says that during the lactation period, the protein content (lactoferrin level) is the highest from the first to the seventh day, after which it rapidly declines. This lowers the percentage of pigs that survive to maturity. Developing sows with a high lactoferrin level would help to increase the survival rate of their offspring, thereby increasing efficiency in production.

After four years of research and trials, the PRIT has successfully generated transgenic pigs harboring a foreign lactoferrin gene. Now, lactoferrin levels are maintained at a high level in the milk for the full 28-day lactation period. The gene has been retained through four generations thus far. The PRIT has proposed a field research and extension program to the COA, and hopes to transfer the technology to the private sector.

In addition, factories are using transgenic animals to produce recombinant protein. The objective of pharmaceutical research is to extract a particular desired material from the milk or blood of the transgenic animals they work with.

Chiang Hoang-yung, manager of the Industrial Technology Information Service, says that at the present stage, genetically modified pharmaceutical products are made using bacteria, yeast, tissue culturing, or recombinant DNA technology. The proteins that microorganism secrete must be processed in order to get active proteins. Use of transgenic animals for production not only avoids this problem, but can increase production volume and lower costs.

Scientists are focusing on pigs, cattle, sheep and other mammals. Of these, they are most optimistic about pigs, because these have larger litters, shorter generation interval, and rapid reproduction.

There have already been successful cases in the Taiwan biotechnology community. A joint project involving the PRIT, professor Cheng Teng-kuei of the Animal Husbandry Department of National Taiwan University, and Dr. Chu Kuang-pang of the Veterans General Hospital, has already successfully produced a transgenic pig with human clotting factor IX cDNA. Wu Shinn-chih says that this material, needed by sufferers of hemophilia B, could in the past only be acquired by purification of human blood; the process was extremely expensive. Today, using transgenic pigs to produce the material, 30 female pigs could all by themselves produce a supply large enough for all the hemophilia B victims in the world. The annual production value would be US$160 million.

Genetic engineering appears to be infinitely profitable and omnipotent. However, it is worth noting that gene transfer has risks: transferred genes can spread to unintended hosts, they cannot be retracted, and they become permanent. If there is a problem, it could very well be difficult to put the genie back in the bottle.

In the future, it is possible that transgenic pigs could be used to produce therapeutic molecules or organs for cross-species transplantation. （courtesy of PRIT）

Science cannot be unlearned

Scholars of ecology are worried that, after millions and millions of years of natural plant and animal evolution, introducing genetic alterations that do not arise from natural laws could destroy the ecological balance.

In fact, this type of accident has occurred before in the US and Europe. Zoologists in Germany have found that transferred genes used to improve crops leak from the host onto bacteria. In England, a case has been discovered of a gene transferred into rapeseed moving onto bacteria and yeast in the intestines of bee larvae. The whole rapeseed field had to be destroyed.

The "manufacturing process" for a transgenic pig. （courtesy of PRIT）

Have you eaten genetic food?

In addition, there is continuing controversy over the safety of genetically modified food. There are concerns that gene transfer foods can produce new toxins or anaphylactogens (sources of allergies).

A scientist in the UK fed potatoes with insect-resistant genes to mice. After an experiment lasting 110 days, not only did the mice develop poorly, they developed problems with their immune systems.

A greenhouse experiment involving ladybugs, conducted in the UK, discovered that, after they had eaten aphids which had fed on genetically modified potatoes, their life expectancy declined by half, and egg production declined by 30%.

You might think that the areas of production of genetically altered crops are mainly in the United States, and so too far away to affect Taiwan. But in fact, genetically modified foods long ago came through our doors. According to statistics, 60% of the soybeans and corn imported by Taiwan are genetically altered. Without our even being aware of it, such ingredients are in the tofu, soybean milk, bread, and cakes we have been putting into our stomachs.

This January, an agreement was signed at a conference convened in Montreal, Canada on the subject of biosecurity. Although the ROC is not a signatory to the treaty, it still needs to follow the norms laid down for genetically modified products.

The two main areas of norms for genetically modified foods at present are safety and labeling. Chen Shu-kong, director of the Bureau of Food Sanitation at the Department of Health, says: "Safety inspection is easy, labeling is what is difficult." Domestic producers feel that labeling will greatly increase their costs.

The Academia Sinica conducted a telephone survey in which 48% of respondents said they would be willing to buy genetically modified food, while 94% said they wanted genetically altered food to be labeled as such. Based on the consumer's right to make informed choices, and on international norms, in the future genetically modified foods should be so labeled. It is urgent to pass the relevant legislation as quickly as possible, and to enforce it.

This gene chip, the size of a postage stamp, has 9600 genes. These can help in research and disease testing. Even intestinal virus chips have been produced to provide to hospitals for clinical tests.

What next?

"The boundary between species has been eradicated. This is progress, but also a source of concern," says Chen Ching-san.

"In theory, modified crops should be harmless, but no one can guarantee that they will be completely without side effects," says Chen. His example is the insect-resistant gene in paddy rice. The toxoprotein produced in paddy rice to fight insects should be harmless to humans. But biological phenomena are complex, so there must be a period of observation, testing, field research, and animal testing before the safety of such rice can be verified.

Regardless, biologists say that there's no point in trying to turn back the clock. "This is a road that we have no choice but to walk. Even assuming there are side effects, the only proper and positive response would be to find ways to address them," says Chen Ching-san.

Man is the latest evolving of the more complex species on this planet. Today, as human technology advances at increasing speed, it seems that we have the ability to make life more "perfect" and "useful." Genetic technology is duplicating the key to biological evolution.

The question is, will mankind secure even richer resources in this way and solve age-old problems like hunger and disease? Or will we create an imbalance in nature that leads to a backlash and an unprecedented catastrophe? Scientists who hold the magic wand in their hands should probably be careful where they wave it.

The decoding of the human genome means another step forward for gene therapy for cancer. Gene therapy research has been going on in Taiwan for many years, and is approaching the stage of testing on human subjects. The upper photo shows a cancerous cell, which reproduces out of control, while the photo below shows a normal cell. （courtesy of Yang Wen-kuang of the Biomedical Sciences Institute of the Academia Sinica）

Gene therapy requires a vector to insert the gene into the cell. The orange colored cells are those in which the gene has already been implanted and begun to function. （courtesy of Yang Wen-kuang of the Biomedical Sciences Institute of the Academia Sinica）

（facing page） The age of biotechnology is just over the horizon. Will this be a new dawn, or a sunset? No one can say for sure.