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Question

how are marker genes removed

Submitted by: Blob


Answer

Expert response from Kyle Scottke, Ph.D.

Molecular Analyst, Bayer Crop Science

Tuesday, 25/11/2014 11:53

Simply stated, a marker gene is removed or segregated away through traditional breeding. If you are interested in the scientific explanation, please keep reading. 

 

Marker genes are an extremely useful tool for generating transgenic (GMO) plants. Although there are several plant transformation methods and many types of marker genes, I will focus on one transformation method and class of marker gene in my answer. Agrobacterium-mediated transformation is one of the most commonly used plant transformation techniques and is used in basic biology research to develop transgenic plants. In this method (as described in the GMO Basics section and in what does genetically altered seed mean), a soil bacteria called Agrobacterium tumefaciens is used to transfer genes into the plant genome. In this system, the DNA sequence that is transferred from the Agrobacterium to the plant is called a transfer DNA, or T‑DNA. The T‑DNA is defined by the presence of two DNA “border” sequences that flank the gene intended to be inserted and allow transfer of the T‑DNA into the plant genome — a process called transformation.

 

Although Agrobacterium has this very useful ability to facilitate the transfer of DNA, it is relatively inefficient, and typically only about 10 percent of plants successfully receive any DNA. This means that only 10 out of 100 plants put through the process will have the gene of interest (GOI) integrated in their genome. That’s where marker genes come in — they allow researchers to identify and focus on plants that have been transformed from among the hundreds or even thousands of plants that are initially generated. This question on GMOAnswers.com about why marker genes are needed might be useful if you would like to learn more.

 

A common class of marker gene used in plant biology is an herbicide-tolerance gene. Using an herbicide-tolerance gene as a selectable marker, researchers can transform a batch of plants and then treat them with herbicide. Plants that do not contain the herbicide-tolerance gene are killed by the herbicide, leaving only the transformed plants. With this in mind, plant biologists can design the Agrobacterium so that it contains two T‑DNA segments, each carrying one gene. For example, one T‑DNA could contain a gene for a trait of interest such as insect control, drought tolerance, etc., while the second T‑DNA could contain a marker gene conferring herbicide tolerance. This strategy is known as a 2 T‑DNA transformation. Important aspects of this 2 T‑DNA transformation system include: 1) when transformation is successful, both T‑DNAs are likely to be inserted in the plant’s genome, and 2) the two T‑DNAs are often inserted into the plant genome separately, likely at different locations within the genome.

 

Since the two distinct insertions of T‑DNA are in separate places in the plant genome, traditional plant breeding can be used to generate plants that carry only the T‑DNA containing the trait of interest, without the T‑DNA that contains the marker gene. This is accomplished by crossing (mating) the transformed plant containing the two unlinked T‑DNAs with a plant that does not contain any inserted DNA to create offspring that contain either, both or none of the T‑DNAs. This is an example of classical genetics (called Mendelian segregation), similar to how you get some genes from your mother and some from your father. In this case, the researcher can identify plants in the resulting progeny that contain only the T‑DNA encoding the useful trait using molecular biology techniques. This is one example of how a marker gene is used to aid in the development of GMO plants. Once plants are identified that carry only the desired T‑DNA insert, these plants will undergo further screening and extensive analyses to demonstrate their safety to consumers and efficacy to growers. 

Answer

Expert response from Community Manager

Friday, 14/11/2014 13:59

In plant biotechnology, marker genes, also frequently called “selectable markers,” are used to identify plants in which a transgenic trait has been successfully inserted. A good example of a selectable marker is herbicide resistance. In this case, the breeder sprays an herbicide on the plants of interest. Those that were not successfully transformed will be affected by the herbicide and discarded. Some marker genes can be both a marker gene and a commercial transgenic trait. In that situation, the marker is never removed.

 

In other situations, markers are removed during the product development process and therefore no longer present in the commercial product. There are a couple of ways to remove a marker gene. 

 

First, if the transgenic trait of interest and the marker gene are delivered in different genomic locations, for example, traditional plant breeding can segregate those plants that contain only the transgenic trait of interest but not the marker gene. The plants containing the marker are discarded and are no longer part of the advancement process. 

 

The second approach would be when the marker gene is being prepared for transformation; it can be flagged through the addition of specific, short DNA sequences in front of and behind it. These sequences are the “beacons” recognized by a special enzyme that will facilitate precise removal of the marker gene later. Most recently, several precision genome engineering tools have been developed that can find and remove the marker gene even without its prior flagging.

 

It is very important to know that in every case where the selectable marker remains in the commercial product, it is treated the same way as the transgenic trait of interest and undergoes the same rigorous safety evaluations. Numerous and robust studies over a span of several years are conducted to evaluate and demonstrate the safety of transgenic plants to human and animal health and the environment.