The targeted insertion of genes and simultaneous removal of the marker gene using site-specific recombination systems

(2005 - 2008) University of Hamburg, Department of Developmental Biology and Biotechnology

Topic

When a gene has been specifically inserted in a plant, it is important to ensure that this transgene is stably expressed in that plant, especially in the case of long-lived trees such as poplars. Otherwise, over time, the plants could lose the characteristic they have gained.

The aim, therefore, is to insert the gene at very specific sites in the genome, ones that have proved suitable for the stable integration of foreign DNA .
In addition, only those gene sequences are to be transferred which are necessary for the desired characteristic. Functional DNA sequences which are no longer required once transformation has been successfully carried out are to be removed again. These include antibiotic resistance genes , for example.

Various systems are available to facilitate the targeted integration of foreign DNA into a plant genome and the subsequent removal of the antibiotic resistance gene used as a marker. One of these so-called site-specific recombination systems is the FLP/FRT system. In this research project it was used to transform poplars and wheat.

Information to methods:

Summary

The FLP/FRT recombination system was successfully used for the first time for wheat and poplars to remove the genetic markers that were no longer required.
Furthermore, a targeted integration of a bar gene into poplars was accomplished using this recombination system.
This, therefore, provides a viable method for producing transgenic plants through the successful elimination of a genetic marker, with subsequent targeted integration of a foreign gene.

Experiment description

As earlier experiments have shown, the FLP/FRT system can in principle be used with poplars. The aim of this project was to develop a practicable method for using the system with poplars and wheat

to remove the antibiotic resistance gene used as a marker
to integrate the "gene of interest" at a specific site in the genome by recombination.

(1) Production of transgenic poplars and wheat. The first stage in achieving these goals was to produce various transgenic wheat plants and poplars which carry an antibiotic resistance gene. This resistance gene lies between the two DNA sequences, which serve as markers for the subsequent targeted excision of the resistance gene and its replacement with the gene of interest (FLP/FRT system).

(2) Removal of the resistance gene. In the second stage the antibiotic resistance gene was removed again by recombination. The process of recombination in this instance was triggered by heat shock, which the plant was briefly exposed to. Then tests were conducted using PCR analysis to see whether the resistance gene has in fact been removed.

(3) Targeted insertion of the transgene. Once the resistance gene has been removed, the aim of the third stage was to integrate the gene of interest at the same site. In this project an easily checkable gene was used (bar gene, resistant against Glufosinate ) which confers herbicide resistance. This recombination was also triggered by heat shock.

Tests were then carried out to see whether the recombination has achieved the desired result. In this case, the plants were treated with the complimentary herbicide of the inserted gene. If they are able to grow under these conditions, they have the herbicide resistance conferred by the target gene: therefore recombination has been successful.

Analysis of the integration site. The integration sites were identified and analysed before and after recombination using various molecular biological methods (PCR and Southern analysis).

Results

The blue colouring in the poplar leaves shows that the antibiotic-resistance gene has been successfully removed. The enzyme of the GUS gene is able to convert a colourless substance into a blue dye.

The control experiment for the recombination was carried out on a wild-type poplar leaf: It showed no blue coloration, since the GUS gene responsible for the colour reaction was not present.

GUS stained transgene wheat leaves. The leaves were havested one day after the successful heat treatment and treated with stain. The blue staining is the proof for a effective removal of the antibiotic resistance gene. above: without heat treatment, below: after heat treatment.

(1) Production of transgenic poplars and wheat: The project began half-way through 2005. Production of the gene constructs that carry the FLP/FRT system and an antibiotic-resistance gene or the target gene (bar) is complete. Wheat plants and poplars were then transformed with these constructs, using a particle gun in the case of wheat and an Agrobacterium in the case of the poplars. Southern analyses have shown that several poplar plants contain the constructs required for removing the transgene/ integrating the target gene.

(2) Removal of the resistance gene: The subsequent recombination to remove the resistance gene was to be triggered by heat shock. Suitable promoters that react to heat were used. Studies in the first year of the project (2006) pointed to a promoter that can be used both in transgenic poplar and wheat plants. It has since been shown that removal of the antibiotic resistance gene could be triggered in poplars and wheat by heat shock. This could be shown through the activation of a GUS reporter gene in the leaves of the poplar and the wheat strains. However, the resulting colour intensity in the wheat lines was considerably weaker than in the aspen (poplar). In addition, a mosaic-like colour pattern was seen on the wheat leaves, indicating that the GUS gene was only activated in certain individual cells. The controls with transgenic wheat leaves without heat shock showed no colour development.

(3) Insertion of the target gene: No wheat line could be generated that contained the gene construct required for the integration. Therefore, further studies were confined to the successfully transformed poplar lines. To activate the FLP recombination system the plants were again subjected to heat shock. After the heat treatment, selection was performed on a herbicide-containing medium. Seven of the eight poplar lines were able to grow on this medium. In these, the gene that mediates herbicide resistance (bar gene) had been effectively inserted in the previously defined gene location. These integration results were also confirmed by PCR analysis.