Techniques of Gene Insertion

Over the past three decades, researchers have developed a number of procedures that improve on the basic methodology of Boyer and Cohen. One area in which progress has occurred involves methods for transferring DNA from a donor organism (or DNA prepared syn­thetically) to a host organism (a process known as gene insertion). Those methods can be divided into two general classes: those that use living organisms (called vectors) and those that use mechanical methods. The three most common insertion methods using vector organisms employ bacteria, viruses, and yeast artificial chromo­some (YAC).

The most common vector used to introduce DNA into plant cells is a bacterium by the name of Agrobacterium tumefaciens. A. tume-faciens is a microorganism that lives in the soil and infects plants, causing a disease known as crown gall disease. The bacterium con­tains a plasmid called the Ti (for tumor-inducing) plasmid, which is altered to include the DNA segment to be introduced into a plant. The DNA segment responsible for crown gall disease is also altered, disabling it so that the bacterium is no longer pathogenic when in­troduced into plant cells.

A second method of gene insertion makes use of viruses. Two common examples are geminiviruses, used to alter the DNA of corn and wheat cells, and caulimoviruses, used to transform members of the mustard family (Brassicaceae), which includes Brussels sprouts, cabbage, broccoli, cauliflower, turnip, and a variety of mustards. Viruses are pieces of DNA encased in a protein shell. They serve well as vectors because they are able to attach themselves to a cell's outer surface and inject their DNA into it. Once inside the cell, the viral DNA takes over the cell's machinery and begins making cop­ies of itself, destroying the host cell in the process. To be used as a vector, two changes in the viral DNA (or RNA) are necessary. First, instructions for viral replication must be disabled so that the virus, once inside the cell, is no longer able to replicate itself. Second, the gene to be inserted into the host cell must be added to the viral DNA.

As its name suggests, the third common insertion vector, yeast artificial chromosome (YAC) is a synthetic plasmid-like structure prepared especially for the insertion of genes into organisms. A YAC consists essentially of three parts: a telomere (either end of a eukary-otic chromosome), a centromere (the central part of a chromosome), and a number of restriction sites at which restriction enzymes can cut. The gene (or genes) to be transferred are inserted into a YAC, which is then mixed with host organism cells. Some of these cells incorporate the YACs, forming a transgenic organism. The YACs' property of special interest is their ability to accommodate very large genes. Their maximum capacity is about 1 million base pairs; by contrast, the limit is of about 10,000 base pairs for plasmids and about 25,000 base pairs for most viruses.

A number of non-vector techniques are also available for inserting a gene into a cell. One approach is to create tiny pores in the walls of host cells by some chemical or physical method, openings that allow genes to enter the cell body more easily. These processes are known, respectively, as chemical poration and electroporation or laser poration. In chemical poration, the addition of some chemical to the host cell causes pores to open in the cell membrane, allowing a gene or genes to flow into the cell's interior. Electroporation accomplishes the same result by administering a brief electrical shock, and laser poration does so by exposing host cells to a microscopic laser beam. As with other methods of gene insertion, scientists do not entirely understand the processes by which genes are incorporated into the cell body, and they cannot reliably predict how many will be taken in or the mechanism by which they will eventually be expressed.

Another method of gene insertion is called bioballistics, or bio-listics. Bioballistics makes use of thin metal slivers that are coated with the genes to be inserted and then fired into the host cell by some mechanism. One such mechanism is the gene gun, shown in the diagram on page 101. The earliest gene guns looked and

Before

-Helium gas

Restraining membrane Kapton disc Microprojectiles Stopping screen

Gas vent

Screw-in < retainers Screen —

Plunger ■

(^^Tissue^^)

After

1^

I     I

Shock wave

© Infobase Publishing

Design of a gene gun

acted much like other types of guns, using gunpowder charges to fire microscopic coated BBs or pellets into a cell. Such guns were generally too powerful, however, and were unable to in­sert genes without destroying the target cells. Today's gene guns are more sophisticated, using compressed helium gas to produce a shock wave that fires coated projectiles into a group of cells. These gene guns fire not only metal slivers but also tiny metallic beads, usually made of gold or tungsten; all such projectiles are coated with the genes to be added to the host cell.

A number of variations on the helium-powered gene gun have been developed. They differ from the original gene guns primar­ily in the mechanism used to accelerate the projectiles into the cell. They use, for instance, centripetal, magnetic, or electrical forces; spray systems; mechanical impulses; shock waves; or electrical discharges.

Finally, for cells that are large enough, genes can be inserted into a cell directly by means of a fine-bore micropipette. The gene to be transferred is first removed from the source with the micropipette. The micropipette is then inserted through the cell membrane of the host cell, and the gene released into the cell body. Under suitable circumstances (which are usually not well understood), the host cell takes up the gene, incorporates it into its own genomic structure, and begins to reproduce it along with its native genes.