Part two of this review will continue to elaborate on the techniques used to modify organisms. The point of these two articles is to present a fairly unbiased view of the strategies and techniques used to identify, isolate, and implant genes for genetic engineering. The conclusion to the two articles can be found at the end of this piece and it is important to remember that these are the words of the author and do not represent A Growing Culture.
Creating the Physical Map: enzymes, PCR, and bioinformatics
The exploitation of polymorphisms for fingerprinting has been widespread, but the information is worthless without a physical map. Labs and Genomics institutes all over the U.S. are using tandem repeats, SNPs, or RFLP to identify unique sequences between individuals. These sequences are easily identifiable by both the scientist and the restriction enzymes (RE), which are a class of enzymes that catalyze the cleavage of DNA at specific sites to produce discrete fragments of DNA. Single base changes, for instance A to G, may introduce a new restriction site into the DNA. These changes are often in the non-coding regions, but are quite common. The polymorphic nature of these non-coding regions, leads to a variable length in fragment after restriction, and these DNA markers are known as restriction fragment length polymorphisms or RFLPs. Again, these segments can help identify differences between individuals.
RE hydrolyze the backbone of DNA leaving a phosphate on the 5’ end and a hydroxyl group on the 3’ end. Each RE has been isolated from bacteria, and their name represents the species they came from, for instance EcoRI from E. coli. They cleave the DNA at a unique sequence known as a ‘palindrome’, where the nucleotide sequence is the same forward and backward. Once the DNA has been incubated with the REs, the methodology of RFLP is as follows: DNA gel electrophoresis to separate fragments, Southern Blotting to immobilize DNA on an inert support, Hybridization between two complementary DNA sequences, and finally the denaturation of the template and the probe readying the DNA for PCR.
Polymerase chain reaction (PCR) is a molecular biology technique to amplify a single or few copies of DNA across several levels of magnitude to ultimately generate thousands or millions of copies of that particular DNA. This is useful because a very small amount of DNA sample can be turned into an amount worth manipulating, studying, or identifying. This is where genomics institutes come in with bacterial artificial chromosomes (BAC) and high information content fingerprinting (HIFC).
Once the sequence of interest has been identified it can be inserted into the Bacterial cell, often E. coli, to be cloned and amplified for sequencing purposes. After amplification, the DNA is extracted from the BAC clones, digested with 5 REs, labeled with 4 unique fluorescent dyes, and placed on the DNA analyzer. At this point the computer takes over with fragment analysis and outputs fingerprinted contigs. Other sequencing such as Automated or Pyrosequencing can also occur. The various contigs, or overlapping sequences of DNA, represent a consensus region of DNA and are the precursors to physical gene distances, gene loci, as well as the template for the isolation of genes. This realm of genetics is known as bioinformatics, and is responsible for every genome map today. With a solid gene map, and a plan of attack, the actual engineering process is now ready to implement.
Genetic Engineering: vectors, bacteria, and some time in the lab
GE technology exists in all fields of science; however, for the purpose of this paper, GE will be in the context plants, especially those used for agriculture. This is also the arena where the most controversy lies; most people are not arguing about GE technology in vaccines, as we are a generally anthropocentric society.
Even though the story truly begins with the advent of agriculture, this story will begin with the vector. Vector in Latin means “carrier”, and in GE, the vector ‘carries’ the desired genes into the cell. The vector in this case, is a plasmid, which is a double stranded DNA ring that has the ability to replicate independently of standard cellular DNA replication mechanisms. There are other vectors such as retroviral, bacteriophage, cosmids, and fosmids, depending on the desired application. In this case the bacterial plasmid is modified by adding the gene of interest, which has been identified by the mapping techniques listed above. This process is not 100% efficient, and after insertion, the bacterial cells must be tested for the receipt of the modified plasmid. This is often done with the addition of a selectable marker, such as an antibiotic into the culture; if the cell has successfully been modified, it will not die when exposed to the antibiotic. The modified bacteria have been identified, and can then be replicated through ‘natural’ cell division. If more than one gene is desired, microarray technology can be used. After the mRNA containing the genes is hybridized they are placed on the microarray slide, essentially a square with a bunch of dots, and scanned with a laser. The result is a multicolored printout; the colors correspond with traits and statistical analysis can occur for better selection. If all has gone as planned, the vector is ready.
The genes have been selected and incorporated into the plasmid (plasmid vectors are owned by companies, and easily purchased for GE), now it is time to insert them into the cell. This can be accomplished through biological or physical methods. The biological methods use viruses or bacteria that naturally infect cells with their own genetic material as part of a survival mechanism. The desired genes are added to the vector; the vector is added to the cell culture, and natural infection occurs. Biolistic particle delivery systems also known as gene guns or biolistics are physical methods that have the ability to inject cells with genetic information. The bullet or payload is a heavy metal (often gold) coated with Plasmid DNA. The payload is ‘blasted’ into the cell with the hopes that a plasmid particle will make it all the way into the nucleus.
These methods require a tissue culture that takes weeks of incubation. Examples of tissues used are: cotyledons, protoplasts, somatic embryos, pollen grains or tubes, ovules, disk leaves, roots, or a callus which is essentially a mound of undifferentiated plant cells. There are also a couple methods that do not require a tissue culture: meristem and pollen transformation. The premise here is the transformation of tissue prior to the differentiation of the germline; the resulting tissue and seeds will be transgenic. This can be achieved through floral dip methods, vacuum infiltration, electroporation, or osmotic shock.
At this point another selectable marker will be used to test if the transgenes were incorporated into the tissue. Plant cells are often exposed to an herbicide such as glyphosate or glufosinate. If the cells die, they were not; however, if they survive they were incorporated and the cells can then be exposed to plant hormones to induce differentiation into the actual plant. There are also other reporter genes such as fluorescents that can be used to identify transgenic success. Whether from a tissue culture or meristematic transformation, the next step will be growing the plant to maturity; using conventional breeding and selection processes to isolate the best strain. Verification of transgene effects should be noticed in T2 and T3 generations before homozygous lines can be selected. At this point the transformation is more or less complete, and the socio political side takes over with regulatory measures. If all regulatory agencies such as APHIS, EPA, and FDA approve the product with their multi-step process, marketing can occur. So where to now?
The Future of GE in Agriculture: thoughts and conclusion
The last century has brought many changes to the face of the earth, most notably in agriculture. Today the average US farmer is feeding approximately 140 people, the highest it has ever been in history, and the highest of any other nation. This can be attributed to dozens of things, and some will say GE varieties of crops are one of them.
Other then the fact that the industry is worth billions of dollars, GE plants arose because genetic modification through conventional plant breeding takes years of selection due to lack of control over how the gene is expressed, combined traits after crossing, high levels of recombination, and an undesirable genetic load. The production of transgenic plants are faster and whether agreed upon by the majority or not, the advent of GE technology in crops, has changed the agriculture landscape forever.
Concerns about GE technology arise all over the planet, to the point where exported U.S. seeds have been burned just to prove a point. But is all of this hatred deserved? Whether a staunch environmentalist or not, understanding the science behind GE is paramount to intelligently discussing the issue. Value-laden debates without understanding are a waste of time, and all people are guilty of this. So after immersing myself in the literature my conclusion is this: GE crops, on a molecular level, are really no different than regular crops and transgenic technology is more or less an accelerated form of conventional breeding and hybridization. Unfortunately, this is only one side of the story, and the situation must be analyzed through a more holistic lense before legislation can take place. Like all human systems, the problem with legislative regulation in the U.S. is corruption. This stems from the fact that many positions on regulatory agencies are filled by previous or current employees of the exact companies that are being regulated!
These companies use devices to “soften” the ecological impact such as antibiotic/herbicide-marker free transgenic plants, chloroplast transformation technology, or terminator technology. The cynic would say that antibiotic/herbicide marker free plants are missing the issue and simply placate regulatory agencies, that chloroplast technology makes sense but may not be 100% effective, or that terminator technology is simply a means for the seed company to stop seed saving in an effort to protect their own patent interests. The advocate for this technology is the farmer who actually has to deal with weed pressure, low yields and a family of five, the employee of the GE company who needs a job, or the ignorant consumer who has no idea about anything and will purchase whatever is cheapest at the grocery story.
Overall, it is clear that the issue is still up for debate, and even though there are dozens of GE plant varieties with excellent traits that improve agrisystems and human health, there have only been a few traits approved for use. These traits such as herbicide resistance and antibiotic exudates where pushed through the regulatory process while improved varieties of rice, cassava, or animal forage still linger in the balance. This is the clear indicator that GE technology is “all about the Benjamins”. If the companies in favor of transgenic plants actually cared about global food insecurity, they would have pushed these varieties through legislation, instead of Roundup Ready® or Liberty Link® technology, which account for billions of dollars in revenue annually. From a strictly molecular level, GE is not different; however, from an ecosystem or socioeconomic view it is, and the implications of this technology as well as the patenting of life should continue to be heavily regulated and scrutinized.