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Q:
I understand that the current generation of transfection vectors are not directed and thus insertion of genes into host genomes is random. I'm curious as to what techniques labs employ to screen genotypes for inserts into coding regions or DNA regulatory units and thus how only those inserts into non coding regions are progressed post transfection.
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A:Expert Answer

For context, it is important to recognize that random genome insertions have been naturally occurring in crops over the ~10,000-year history of agriculture.  In some crops, more than 90 percent of the genome consists of these types of random insertions. It is worth noting that during this long period of time, these “modified” crops have been consumed safely by humans and animals, and there is a very long history of safe use of crops with this type of modification.

 

In the case of GM crops, you are correct in saying that the current methods (transfection systems) we use to insert a gene into a plant typically do not result in a targeted insertion either, but you need to consider the rest of the total process. Researchers use a number of characterization techniques to understand and locate exactly where the gene was inserted, and subsequently select only those plants with the best insertion points (optimal molecular profiles) for further work.

 

Unlike new crop varieties developed by other breeding methods, researchers fully characterize GM crops at the molecular level. Following is a more technical description:

 

  • For any given “event” (events are the individual products of transformation), researchers will assess the number of distinct insertion sites resulting from transformation using a variety of molecular biology techniques (e.g., genome sequencing, PCR based methods or Southern blotting). We typically consider the events that contain a single insert as optimal, and identify and eliminate others from further work.
  • For any insertion site, researchers can determine the DNA inserted and its location in the genome through sequencing, which involves determining the precise genetic sequence of A's, C's, G's and T's, in an event’s genomic DNA. By examining the inserted DNA, we can confirm that the insertion occurred as intended. Also, by examining the genome sequence surroundin,g or “flanking,” the insertion site, we can tell if any native coding or regulatory regions have been disrupted. Once again, only events with the intended insertion and without disruption of native sequences are kept for further work.
  • Given this full characterization of the number and nature of insertions produced during transformation, we are able to identify and keep only the desirable events, and exclude all others.
  • In addition, during the development of new varieties, a researcher will screen thousands of plants, based on their appearance, to make sure there are no apparent unintended effects.  However, researchers do this for all new varieties, whether they are being developed by GM technologies or through conventional breeding techniques.

 

Last, I’d mention that even though researchers fully characterize GMOs down to the level of their DNA, similar types of insertions have happened in nature many, many times over the history of cultivation and consumption of crops.  Those resulting crops have remained safe for consumption in the context of all of these changes.

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