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Microbial recombination systems

Controlled gene excision

Recombination systems using bacteria and yeasts are being used successfully to remove marker genes from the genome of transgenic plants. They can also be used to remove transgenes from pollen, thereby preventing the transgenes from spreading in the environment.

Recombination is one of the causes of genetic variability of organisms. It is a natural process in which DNA segments are exchanged and rearranged. One way that recombination takes place in bacteria and yeasts is through recombination systems. These consist of two components:

  • Recombinase, anEnzym.
  • Certain recognition sequences, known as ‘sites’: The recombinase recognises ‘its’ sites and cuts a DNA sequence flanked by them out of the DNA strand. If such sites are also found at another location on the genome, the recombinase can integrate the DNA sequence again at this location (homologous recombination)..

Several naturally occurring recombination systems are now known to exist. Three of them have been, or are being, used in biosafety research projects:

  • The Cre/loxP system originates in the P1  bacteriophage. It consists of the Cre recombinase and the lox sites.
  • The resolvase/res system (another bacterial system). The cutting enzyme in this case is a resolvase. The corresponding sites are res sequences.
  • The FLP/FRT system comes from a yeast. In this case, the recognition sequences are the FRT sites, which are recognised by the FLP recombinase.

In the methods described here, the recombination systems are used only to excise selected DNA segments. The excised DNA is not reintegrated in the plant genome at another site because there are no recognition sequences, and is instead broken down by enzymes within the cell itself.

Recombination systems for eliminating marker genes: First, transgenic plants are produced with a marker gene flanked by the sites. If the marker gene is to be removed again, the recombinase gene is also introduced into the plants and the marker gene is cut out.

There are two alternative methods for inserting the recombinase into the plant and removing it again:

  • The recombinase gene is expressed only temporarily in the plant (transient gene expression), without being integrated in the plant genome. In this method, the plants are infected with genetically modified Agrobacteria or viruses that carry the recombinase gene. Viruses are eliminated during seed formation and Agrobacteria are not passed on to progeny at all.
  • The recombinase gene is transferred to the plant genome together with the marker gene flanked by the sites and is cut out again along with the marker gene. For this method, the gene has to be linked to a specific promoter that controls when the excision takes place.

The Cre/loxP system has already been successfully deployed in biosafety research projects to eliminate marker genes in tobacco, sugar beet, potatoes, oilseed rape and grapevines. The conditions in grapevines still need to be improved.

A completed biosafety research project was able to demonstrate the functionality of the resolvase/res system in potato protoplasts. It also optimised the system for the specific conditions found in potato plants.

Another completed biosafety research project successfully used the FLP/FRT system to eliminate marker genes in poplars and wheat.

Recombination systems for eliminating transgenes from pollen (biological confinement). This method is similar to marker-gene elimination, except that in this case, the sites flank the target gene (the gene that expresses the desired trait in the plant).

The recombinase gene is inserted into the plant genome along with the target gene and the marker gene, after being linked to a promoter that is only active in developing pollen. This means that the recombinase gene is expressed only in the pollen and it is only here that the target gene is cut out of the genome. The vectors are designed in such a way that the marker gene and recombinase gene are cut out at the same time.

This results in a transgenic plant that forms transgene-free pollen, and is designed to prevent transgenes spreading in the environment through outcrossing.

Two current biosafety research projects are using the Cre/loxP system and the FLP/FRT system to remove transgenes from the pollen of poplars and maize.

Using recombination systems for gene targeting: Another key application of recombination systems is gene targeting. Here, recombination systems are used to insert DNA sequences rather than to cut them out. The recognition sequences for a recombinase are inserted into a plant genome and the integration site is first characterised using molecular genetic methods. Then the recombinase can be used to integrate any gene of interest into the plant genome in a targeted manner. Gene targeting can be combined with the removal of a marker gene.