The targeted insertion of genes by gene targeting

(2005 - 2008) University of Karlsruhe, Botanik II

Topic

A DNA break can be specifically created in the plant genome using a specific restriction enzyme called I-Sce I, and can be subsequently closed again by means of homologous or illegitimate recombination . As was demonstrated in the precursor project, this technique can be used e.g. to remove marker genes from the genome of transgenic plants.
Using homologies in an existing DNA section, DNA can be specifically inserted at this site in the plant genome (gene targeting ).

The aim is to combine both approaches: the targeted integration of genes at a specific site in the genome and removal of the marker gene. A further aim is to establish an efficient system that can be applied to any crop variety. Initially, however, the system is being tested on two crops, tobacco and Arabidopsis thaliana .

Precursor project:

Targeted insertion of genes at specific sites in the plant genome

Further information on this procedure:

Summary

The aim was to establish a system for the model plants Arabidopsis thaliana and tobacco which would enable the targeted integration of genes into the plant genome . The experimental strategy involves excising in vivo a linear DNA molecule (donor sequence) from the plant genome using highly specific molecular scissors and identifying its site-specific integration (gene targeting) at a known target sequence. In the experimental system used, these gene targeting events would lead to the formation of an intact kanamycin‑resistance gene , enabling plants to be selected in which the desired reaction had occurred.

The linear DNA sequence was successfully excised in vivo from the genome of both plants and for Arabidopsis thaliana in particular the technique proved to be extremely efficient. This shows that it is possible to excise DNA sequences very successfully from the genome of living plants. However, no kanamycin-resistant plants were obtained from either crop under the experimental conditions chosen. Consequently it was not possible to identify the targeted integration of a homologous DNA sequence before the project came to an end.

As a result of the very promising results concerning the excision of the donor sequence from the Arabidopsis genome, further studies will be carried out using a modified experimental strategy.

Experiment description

The aim is to integrate a gene sequence, the targeting sequenz, into an existing gene sequence in the plant, the target locus, using gene targeting. The targeting sequence is the sequence between the two I-Sce I cutting sites. To test the system, the targeting vector in this case carries part of the kanamycin resistance gene (KA) within the cutting sites. Outside each of the cutting sites there is also one part of the GUS gene (blue arrow), which is important for the subsequent colour reaction. The target locus is the selected integration site in the plant genome and carries the second part of the kanamycin resistance gene (red arrow, NA).

To integrate the targeting sequence specifically at the target locus, the following steps are carried out both with tobacco plants and with Arabidopsis thaliana:

(1) Producing independent transgenic plant strains. Initially the targeting vector and the target locus are transformed in two different plant strains:

One plant strain carries the targeting vector, which is surrounded by a colour marker, the GUS gene. This can be used to subsequently test whether the targeting vector has been successfully excised from the genome of the transgenic plant.
The other plant strain carries the target locus. The restriction enzyme I-Sce I, which later becomes active in the generative tissue of the plants to facilitate gene targeting, is transferred at the same time.

Molecular biological techniques are used to select plants from both plant strains which have integrated the relevant transgene only once in their genome.

(2) Crossing both plant strains. The two plant strains are crossed with each other. Some of the offspring are sown on soil containing kanamycin and tested for their resistance to it. Plants that survive on this soil have the resistance gene. Gene targeting has been successful with these plants.

The remaining plants are sown on a medium without kanamycin. A colour reaction is used to determine which plants have successfully excised the targeting sequence and repaired the GUS gene.

A comparison between the number of plants from which the targeting sequence was successfully excised and the number of plants in which the kanamycin resistance gene was successfully restored provides information about the effectiveness of the gene targeting system.

(3) Testing the sites of homologous recombination. The plants in which the kanamycin resistance gene has been successfully restored will undergo further molecular biological examinations to find out whether the targeting sequencehas been fully integrated at the target locus.

Results

(1) Producing independent transgenic plant strains

In the first year of the project (2005) the targeting vector and targeting locus constructs were produced. Agrobacteria were used to insert them into tobacco plants and Arabidopsis thaliana. The constructs were identified in the first generation of progeny of both organisms.

The following year (2006) more plants were transformed. The seeds from these plants were cultivated and selected until the transgene in question was present only once in the genome. For tobacco, six lines were identified with the targeting vector and five with the targeting locus. In Arabidopsis each construct was found in four lines.

The blue colouring of the young Arabidopsis plants (approx. fourteen days old) indicates successful recombination. The targeting sequence has been cut out, thereby repairing the GUS gene involved in the colour reaction

Offspring of Arabidopsis crosses on kanamycin selection medium.

Cell culture of Arabidopsis leaflets. The leaves died in three of the potential gene targeting strains under investigation.

Diagrams and photographs: Prof. H. Puchta, University of Karlsruhe

The gene with the I-SceI coding sequence was then inserted into all the targeting locus lines. In Arabidopsis the gene sequence was placed under the control of three different promoters . In tobacco, I-SceI constructs with two different promoters were used. These newly transformed lines are currently being tested to see whether the I-SceI coding sequence is present only once in the genome.

In parallel with this, an inducible promoter system was tested in the targeting locus lines of Arabidopsis and tobacco. For Arabidopsis, however, it was shown that this promoter triggers expression of the I-SceI gene even without any external influences. The system was therefore discarded for gene targeting purposes.

(2) Crossing both plant strains.

In the third year of the project (2007) the tobacco and Arabidopsis plants containing the targeting vector were crossed with plants containing the target locus construct and the molecular scissors I-SceI.

For the progeny of the crossed Arabidopsis strains it was shown that the targeting sequence had been cut out of the targeting vector very efficiently; this was shown by the widespread blue colouration, which in some cases affected entire leaves. In contrast, only small areas of blue were detected in the tobacco experiments, suggesting that the excision of a linear donor sequence in the tobacco genome is a limiting factor.

The plant strains were propagated by selfing (self-fertilisation) and seeds from the subsequent generation were placed on a kanamycin-rich selection medium. Numerous seedlings were identified growing on this nutrient medium, all of which were examined using molecular-genetic methods. Surprisingly, no gene targeting events were found either in Arabidopsis or in tobacco. One possible explanation could be that, although site-specific integration occurs, leading to the restoration of a functioning kanamycin-resistance gene, its expression is too weak to enable selection at seedling level.

Therefore, to selectively increase the number of potentially kanamycin-resistant cells, the experimental strategy for both crops was changed. Plants strains from crosses containing both the targeting locus and the targeting vector were transformed with an I-SceI construct and propagated over two generations using appropriate selection steps.

Leaflets were then cut off and transferred to the cell culture medium. The modified conditions did not increase the efficiency of excising the targeting sequence in tobacco and no kanamycin-resistant cell cultures were obtained. Consequently work on tobacco ceased.

Since the excision of the linear targeting sequence is not limiting in the model plant Arabidopsis, further investigations will be carried out even after the official completion of the project to find indications of increased gene targeting efficiency by further modifying cell culture protocols.