How to introduce a transgene into a plant cell?
There are several techniques to introduce a transgene into a plant cell. This can be done by gene bombardment, electroporation or by using a technique invented by nature: Agrobacterium tumefaciens mediated gene transfer.
Integration site of the transgene into the plant genome.
Once the transgene enters the nucleus it can be integrated into the plant genome. The transgene becomes integrated at random positions in the genome, in the different chromosomes, in and between genes. This is a big disadvantage because important genes can be disrupted, every transformed plant cell gives a different result, etc…
Experiments showed a relationship between the place of transgene integration and double strand breaks in the plant genome. A double strand break in the genome is a normal phenomenon and can be repaired by special enzymes. In an experiment with tobacco plants, a double strand break at a specific place was induced with an endonuclease. When the transgene was brought into the nucleus, the transgene was mostly integrated at the place where the double strand break was induced (1).
Inducing double strand breaks at specific location.
By using this knowledge, scientists try to control the transgene integration site, this is called genome engineering. The integration site can be controlled by inducing a double strand break at a very specific place in the plant genome. To realize this, two things are needed.
1) an enzyme that can cut the DNA strand. This enzyme is called an endonuclease, and
2) a molecule that can recognize a specific DNA sequence and bind to it. The endonuclease is fused to this molecule, so the whole thing becomes a target searching and cutting machine.
There exist several molecules that can recognize and bind to a specific DNA sequence. One example is the TAL effector. This is a protein made by a bacteria, the Xanthomonas bacteria. The bacteria excrete this TAL effector into a plant cell. The TAL effector will go to the nucleus and bind to a specific DNA sequence. The TAL effector has also a gene activating domain. The specific DNA sequence that is the target of the TAL effector belongs to genes that suppress the plant defense.
Scientists are trying to make synthetic TAL effectors, but without the gene activating domain and replace it with an endonuclease. This is called a TALEN. By modifying the protein, it is possible to change the target DNA sequence (2). Other molecules that can bind to specific DNA sequences are Zinc-Fingers and are also used for specific transgene insertion (3).
Possible problems/challenges for the future.
If scientists want to use the TALEN nucleases, or Zinc-Finger nucleases, for inducing double strand breaks into the genome they first have to insert the nuclease enzyme into the nucleus. This is not so easy to accomplish. One can choose to insert it as a transgene that will integrate at random in the genome and translate for the nuclease. A second transformation is needed to bring in the transgene of interest that will be most likely be inserted into the induced double strand break.
It is also possible to transiently express the TALEN enzyme by inserting it as a RNA molecule together with the transgene of interest.
Unfortunately, the efficiency is still very low and research will continue to find better solutions.