‘Don’t judge a book by its cover’: the science behind genetic engineering

In the last two posts, I addressed the topics of DNA and natural genetic variability. Here I’d like to use those topics as a starting point for a discussion about the technology behind genetic engineering. You probably recognize the saying “don’t judge a book by its cover”. While this saying is applied to people and, well, books, I think it is also a good sentiment to consider when looking at genetic engineering. For nearly 50 years, scientists have been transforming organisms through genetic engineering techniques and studying the effects of such changes. To date, scientists have thoroughly examined possible concerns regarding genetic engineering, finding nothing that isn’t seen elsewhere in natural systems. So today, why are we still surrounded by such negativity and fear about genetic engineering? I’d say it’s because people are judging the book by its cover – believing statements for which there is no supporting evidence. With people, we cannot make an accurate judgment of their character before we know them, and the same goes for the products of genetic engineering—in order to make educated choices for ourselves and for future generations, we must understand how and why genetically engineered plants are made, starting with how the technology works.

So, how does genetic engineering work?

As with all modern technology, genetic engineering has changed greatly since its initiation. It has become increasingly accurate, efficient, and refined, and improvements in other areas of science and technology have permitted comprehensive analysis of all possible effects on the resulting plants and products. Here I’d like to give a brief summary of some milestones in the history of genetic engineering and how these technologies work. To keep this post from being too long, these descriptions will be slightly summarized, so if you find you have additional questions or would like more information, please don’t hesitate to let me know!

Tissue Culture

Almost all methods of genetic engineering rely heavily on tissue culture, a technique that was established in the early 1900s. Tissue culture is the growth of living cells in an artificial medium, usually composed of water, agar, nutrients, and compounds, such as natural hormones. In plants, tissue culture cells are often obtained by cutting pieces of leaves or stems and placing them on an artificial medium containing specific hormones. These hormones, and other compounds, encourage new, undifferentiated cells to grow. These cells have the potential to grow into a new plant, a characteristic called totipotency. To encourage the cells to grow into plants, the tissue culture recipe can be adjusted to contain different levels of the necessary growth hormones and nutrients. Eventually, whole plants can grow from single cells. Tissue culture techniques vary for different species of plants and the methods have been refined for nearly one hundred years, with new advancements still being made today. Tissue culture is used for plant propagation (generating more plants of a specific cultivar), as well as for genetic engineering. In genetic engineering, the DNA of a single cell is altered through various techniques and then grown into a new plant, where all of the cells will have the altered DNA. The following addresses some transformation techniques:

tissue-culture-series
Tissue culture steps. Far left: leaf tissue cut from a mature leaf, placed on tissue culture medium. Center left: new cells develop from the leaf tissue and form a callus (a mass of undifferentiated cells). These cells will be used for genetic transformation. Center right: the cells divide and begin to form new shoots. Far right: small plantlets have developed. *at each step, the tissue is transferred to new tissue culture medium, often with different concentrations of hormones and nutrients.

Induced Mutation

Initial methods of genetic engineering were through induced mutations. Scientists would expose a cell to certain sources of radiation, causing DNA mutations to occur. In this method, scientists have no control over what genes are being mutated and what effects this will have on the cell and eventually the whole plant. For this reason, induced mutations are not commonly used for crop improvement purposes but rather for the discovery of new genes. This works because scientists can look for individuals with unique characteristics and then compare the mutated DNA with that of a non-mutated individual, thereby discovering the gene that controls the trait.

Transformation Technology

The next advancement was transformation technology, or the ability to add a specific gene to a cell. This was achieved in 1972 when the first recombinant organism was created. A segment of DNA known to produce antibiotic-resistant proteins was inserted into a bacterial cell. The cell combined the foreign DNA sequence with its own DNA and therefore became capable of producing the protein. This discovery eventually led to the production of human insulin from bacterial cells in 1982, when the human insulin gene was incorporated into bacterial DNA, technology that is still used today to produce insulin.

With transformation technology, scientists can introduce a gene of interest into a tissue culture cell through several techniques, two common ones being agrobacterium and biolistics.

agrobacterium-tumefaciens-on-horse-chestnut-4
Natural Agrobacterium gall on a horse chestnut tree

 

Agrobacterium is a soil bacterium that infects plants in nature. When it infects a plant, it inserts its DNA into the plant’s DNA and causes the plant to form a gall (or tumor) for the bacteria to grow within. By inserting a gene of interest into the agrobacterium cell and infecting tissue culture cells with the transformed bacteria, the gene of interest can be inserted into the plant DNA and subsequently expressed in the plant cells that grow.

When using biolistics for transformation, microscopic gold particles are coated with the gene of interest and are shot into young plant cells using a ‘gene gun’, typically a hydrogen gas-powered particle delivery system. The DNA on the gold particle enters the cell and is incorporated into the plant cell DNA, just as in the agrobacterium transformation.

gene-gun
Schematic of a biolistic particle delivery system

In both methods, the cells will be screened to select those that were successfully transformed (containing the new gene), this is done using selectable markers. A selectable marker is a gene, that is included in the DNA segment containing the gene of interest, which codes for a specific trait that scientists can test for. In plants, it is often a fluorescent protein, a protein that turns blue when stained or an herbicide resistance gene, which allows cells with the gene to be easily identified. Scientists will keep the transformed cells and grow them into whole plants so they may study them and see the effects of the added gene.

Unfortunately, there is no way to control where within the plant DNA the new gene has been inserted, so scientists examine all aspects of the transformed plants to determine how many copies of the foreign DNA were inserted, where they were inserted, and how they affect plant growth and function. Sometimes concern is raised that the added selectable marker genes, for fluorescence or herbicide resistance, for example, are being expressed in genetically engineered plants; however, through traditional breeding, these genes are selected against and after one generation the resulting transformed plants will contain only the gene of interest.

Intragenics and Cisgenics

Oftentimes, the gene of interest being used in a plant transformation is derived from bacterium DNA or that of another plant or non-plant species; however, in some cases, a gene can be used from the same plant species or a compatible relative, known as intragenics and cisgenics, respectively. Essentially, this means that it is biologically possible for the gene of interest to be transferred to the host plant through traditional breeding practices, however, traditional methods have proven unsuccessful or inefficient, so the gene is inserted through transformation instead. Even though the processes are the same and the molecular composition of the DNA is the same regardless of what species of organism it originated from, plants genetically engineered using intragenics and cisgenics tend to have fewer regulations than those engineered with a non-related species’ gene.

Targeted Gene Editing

The most recent advancement in genetic engineering technology is the use of endonucleases to facilitate targeted gene editing. Endonucleases are enzymes that exist naturally in the cell and make breaks in DNA molecules. Scientists can design endonucleases that will bind to specific DNA sequences and make a targeted break at that location, which means they can make a cut in a specific gene or at another exact DNA location. When the break is made, it may be repaired by reattachment of the cut ends. When this occurs, it is common for some nucleotides at the break in the DNA sequence to be lost, resulting in a nucleotide deletion and loss of function for that gene—known as ‘gene knockout’. Alternatively, because scientists know where the endonuclease will generate a break, they can construct a DNA sequence of the gene of interest with nucleotide ends that match those at the break. Then, after the cut is made, the original DNA will repair itself by inserting the new gene sequence in the break—known as ‘gene replacement’. There are a few slightly different technologies that have been developed to conduct targeted gene editing: ZFN (zinc finger nucleases), TALENS (transcription activator-like effector nucleases), and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats, with the Cas9 protein).  The most important feature of these techniques is that, much like editing a word document, they allow scientists to precisely edit the DNA sequence, changing only one or a few nucleotide bases as a very specific location in the genome, and because the nucleases exist naturally in cells, they are degraded by the cell once the genome edit has been made, leaving behind only the edited gene DNA sequence.

cas9_green-01-1024x1024-400x266
Steps of targeted gene editing, CRISPR/Cas9 shown. Image: igtrcn.org

Takeaway points:

  • Scientists have conducted genetic engineering for almost 50 years, giving them time to improve upon the techniques used and thoroughly evaluate the resulting genetic changes that occur in the bacteria, plants, or animals, that they’re studying.
  • Genetic engineering techniques make use of naturally occurring processes to introduce new DNA into a cell, be it soil agrobacterium or endonucleases.
  • Cells are transformed while in tissue culture and then grown into mature plants. Scientists can then study the altered plant genetics for several generations through traditional breeding methods. Primary research is always conducted in controlled environments, never in field settings until the effects of the genetic change are well understood.
  • Scientists study any possible change that may have occurred in their host organism as a result of genetic engineering, and consider all conceived risks associated with their specific genetic change. If a less desirable trait has unintentionally arisen from their efforts, the transformed plants are discarded and a new approach is taken to ensure the stability and safety of the resulting plants.

Hopefully, this summary of genetic engineering techniques provided new insights toward plant biotechnology and why it’s important to understand the science of genetic engineering rather than ‘judging the book by its cover’. The following posts will address the reasons why plants are genetically engineered as well as which crops are genetically engineered and for what traits.

 

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