When planning a genetic engineering project, scientists work out the molecular details of the GMO they intend to produce. These details include identification of DNA sequences encoding the desired trait, choice of marker genes, and nature of regulatory sequences that will direct expression of the transgene. Choices are also made regarding minimization of extraneous DNA, options for targeting the site of insertion, as well as the method of transformation.
Transformation methods for inserting new genes into plants are relatively inefficient; only a very small proportion of treated cells actually take up the new DNA. Marker genes are included in the segment of inserted DNA in order to distinguish cells that contain the new genes from those that do not. Some marker genes encode enzymes that lead to the production of a pigment or fluorescent light, allowing easy identification of GM cells. Other marker genes encode proteins that inactivate antibiotic compounds; when treated cells are grown in the presence of the antibiotic, only those that took up the new DNA are able to survive. In the past, the gene encoding neomycin phosphotransferase II (nptll, the so-called "kanamycin gene") was the preferred marker because it provided a cheap and effective way to grow selectively only the GM cells.
Concern arose that GM plants containing antibiotic-resistance genes would, if consumed as food, present a risk to individuals taking the antibiotic as a therapeutic agent. Despite numerous detailed studies that unanimously concluded the risk was immeasurably low, and despite approval by the food safety regulatory agencies in numerous countries, public opinion remains opposed to the presence of antibiotic-resistance marker genes in foods. In response, developers of GMOs to be used as food are moving away from these genes. Ongoing efforts are under way to identify other types of genes useful as markers and to develop methods for removing marker genes before GM products get to the market. It is worth noting that even though the protein encoded by the nptII gene presents negligible biosafety risk, GMO designers are well advised to consider such concerns.
Molecular biologists have identified a number of promoters able to turn on gene expression in specific tissues. In plants, these tissue-specific promoters restrict transgene expression to roots, leaves, or other selected tissues where the new protein is desired. For example, a leaf-specific promoter, directing toxin production in the leaves but not roots, stems, or flowers, could control a gene encoding a toxin active against a leaf-attacking pest. In transgenic animals, a tissue-specific promoter has been used to direct transgene expression in mammary glands so that the new protein is secreted in milk.
Inducible promoters can switch transgene expression on and off during the life of the plant. For example, certain promoters respond to a chemical signal; simply spraying the transgenic plant or plant part with that chemical can activate them. Water stress, temperature, mechanical damage, light, or various other types of stimuli activate other inducible promoters. The next generation of GMOs is expected to make use of these more sophisticated gene regulatory sequences that can contribute to reducing potential risks.
Cells transformed by Agrobacterium-mediated DNA transfer methods usually contain, in addition to the desired gene or genes, extra pieces of DNA that come from the Agrobacterium vector. Although vector-derived sequences rarely cause any problem, one view holds that the safest approach to design ing GMOs is to avoid including any extraneous DNA sequences. An alternative approach, direct gene transfer via a "gene gun" or electroporation, avoids the potential for inserting unnecessary vector DNA because no vector is used. Other transformation methods make it possible to insert transgenes into chloroplast DNA. The value of this approach is that pollen grains of most, but not all, plant species do not contain chloroplasts; therefore, concern about the spread of transgenes via pollen (gene flow) is essentially eliminated.
Although these methods of advance risk management are easy to implement, they must be integrated into the research plan before the first candidate GMOs are produced. If applied, they simplify later risk assessment by avoiding certain features known to raise questions of risk.
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