How are genes engineered?

If you are uncertain about what is a gene, we recommend our elementary level introduction "What is genetic engineering?". Below we will use a minimum of technical terms.

Contents

• Introduction
• Methods for gene insertion
• Bacteria as gene carriers
• Viruses as carriers
• Mechanical methods
• Tedious and expensive
• The problematic "insertion package"
• Markers
• Promoters
• DNA designed to ensure gene insertion
• Unpredictable
• Conclusion

Introduction

A gene consists at an average of about 3000 "code syllables". So it is a fairly large piece of information that is to be transferred.

1. The desired gene is taken out of the DNA of the donor cell. Special techniques are used, that are of no imporance here.

2. A gene is much too small to be inserted with some kind of microsurgery. Therefore some carrier ("vector") is required which takes the gene into the recipient cell and somemhow gets it inserted into the DNA of the recipient.

Methods for gene insertion

Different means are used for carrying the desired gene into the hereditary substance.. For plants, the most common method is the use of a bacterium, AgrobacteriumTumefacsiens. This method is however not usable for cereals. For those, insertion with the "gene cannon" or microinjection is used. For animals, certain viruses are used. These methods will be described briefly below.

1. Bacteria as gene carriers

A bacteria with the name Agrobacterium Tumefasciens has the ability to induce a kind of benign tumor growth in the plant it infects. This it achieves by inserting some genes into the DNA of the plant. In genetic engineering, the gene that is to be inserted is hooked on to the bacterial DNA. Then the recipient cells are exposed to the bacterium, so that they become infected by it. The bacterial DNA with the engineered gene is then inserted into the DNA of the recipient.

2. Viruses as gene carriers

Viruses are pieces of DNA. Some are encapsulated within a protein shell. Their DNA enters the cell at infection and goes to the cell nucleus. There the virus DNA forces the cell to make many thousands of copies of the virus. The viruses used for gene transfer are modified so that they will not induce virus copying in the cell. They will introduce the desired DNA into the DNA of the recipient in similar manner as the Bacterium tumefasciens. Along with it some virus genes also are inserted.

3. Mechanical methods

The gene cannon

It uses very small golden beads as cannon balls. The desired gene is "glued" on to the surface of the ball. Thereafter the ball is shot into the cell nucleus.

Microinjection

A solution with the desired gene is injected into the cell.

Tedious and expensive

The insertion methods have all in common that it is impossible to control where the gene will attach . It is a completely random procedure. So the inserted DNA can be hooked on to the recipient DNA anywhere. It is a sheer matter of luck if it happens to stick in the very small proportion of DNA that is active. And even then, there is a risk that the insertion will have occurred in such a place that the result is a disfigured, weak or disease-prone organism (which has been repeatedly observed to be the case).

Therefore genetic engineering is a tedious and very costly procedure. Only a small proportion of all the attempts will result in an organism that seems "substantially equivalent" with the natural counterpart. But even then, it may have a chemical abnormality that is hazardous to the health of those eating it. The trouble is that there is no fully reliable method to detect dangerous substances. And the existing methods for safety testing are very costly and time consuming.

This is why the biotechnology industry has taken great pains to make the regulators to regard genetic engineering as just a variation of breeding. If so, they can accuse the authorities for unjust discrimination of their products. - If food from conventionally bred organisms must not be tested, why should food from organisms bred through genetic engineering have to be tested is their argument. Unfortunately, in the first round, the industry has been successful with this strategy. They have been able to make the regulators believe that there is no important difference between genetic engineering and conventional breeding. This claim has however no scientific basis, see "Conventional breeding is fundamentally different from genetic engineering". And most importantly, the differences are so important that consumers will be exposed to potentially serious health risks if they are neglected.

The problematic "insertion package"

The proponents of biotechnology seldom mention that the desired gene is very seldom inserted alone. To make the insertion successful and effective, other genes, taken from various organisms, including viruses, plants or bacteria need to be added not. In addition to the carrier genes mentioned above there are other important categories of DNA that commonly are added in the "insertion package". These are presently marker genes, promoters and genes promoting the "penetration" of the species barrier.

Markers

Even if the gene package has become successfully inserted, it may attach to a region of non-active DNA. Then the gene marker will be silenced and the cell will not survive antibiotics. So only genes that have been inserted into active regions, will express antibiotics resistance as well as the properties conferred by the Trait Gene.

There is no way of seeing if the insertion has been successful. Therefore some "marker gene" is used, that is inserted along with the desired gene.

The success of gene insertion is tested by adding the antibiotic to the culture of recipient cells. The idea is to find out which ones are resistant to the antibiotic. So the cells that survive the exposure to the antibiotic are those that carry a successfully inserted resistance gene. Then, most probably, the desired gene also has been inserted.

The most common marker so far has been a gene that confers resistance to Kanamycin, an antibiotic that belongs to a group of valuable antibiotics. Even if it is itself not used much today, it is closely related to valuable antibiotics. And there is a tendenency for cross resistance. That is resistance to Kanamycin may also confer resistance to its relatives. Another marker, used a/o. in Novartis Basta Corn is a gene that confers resistance to Ampicillin, a valuable and commonly used antibiotic.

It is suspected that the antibiotic gene markers might lead to an increase of the numbers of bacteria that are resistant to the antibiotic.

Promoters

Even when the gene package has been inserted in an active region, the Trait Gene may express the desired trait only weakly or to a varying degree (that is, the production of the corresponding protein is not stable and high).Therefore, to ensure that the Trait Gene will express the trait persistently, a promoter is added to the insertion package.

Promoters are natural parts of chromosomes. Their function is to enhance the activity of a certain gene. Normally these are under the control of regulator genes that turn them on and off. But in genetic engineering the corresponding regulators are normally not present as the promoter comes from an other species. This means that the promoter will exert a strong and persistent stimulating influence on the Trait Gene. This is basically unnatural as normally the activities of all genes are regulated in response to the conditions in the cell. It means that the fine tuned cellular feed-back balance system is persistently disturbed in this respect.

The promoter commonly used in genetically engineered plants is the Cauliflower Mosaic Virus (CaMV) promoter. It is a strong promoter that has a high species compatibility. A potential problem however is that the virus DNA of the promoter may combine with infecting viruses so as to give rise to new viruses, see "New viruses may be created in GE-crops".

Gene package promoting gene insertion in a foreign species

The cell has the ability to identify genes that are alien to the species. There are strong mechanisms that prevent such alien genes from combining with the genome. They break down foreign genes, or prevent their replication, or cut out and inactivate a foreign gene that has been able to insert itself. This is called the "species barrier".

This barrier is a major problem in genetic engineering. To overcome it, gene technology has invented various combinations of genes that have the ability to promote penetration of the species barrier. They consist of genes from viruses or bacteria that faclitate the insertion of foreign genes into the DNA of a new host.