Neomycin phosphotransferase (npt) gene
Two neomycin phosphotransferase genes are used in selection of transformed organisms: the neomycin phosphotransferase I (nptI) gene and the neomycin phosphotransferase II (nptII) gene. The second one is the more widely used. It was initially isolated from the transposon Tn5 that was present in the bacterium strain Escherichia coli K12. The gene codes for the aminoglycoside 3′-phosphotransferase (denoted aph(3′)-II or NPTII) enzyme, which inactivates by phosphorylation a range of aminoglycoside antibiotics such as:
- geneticin (G418), and
NPTII is probably the most widely used selectable marker for plant transformation. It is also used in gene expression and regulation studies in different organisms in part because N-terminal fusions can be constructed that retain enzymatic activity. In animal cells, G418 and neomycin are used as selectable agents.
NPTII protein activity can be detected by enzymatic assay. In other detection methods, the modified substrates -the phosphorylated antibiotics- are detected by thin-layer chromatography, dot-blot analysis or polyacrylamide gel electrophoresis.
Plants such as maize, cotton, tobacco, Arabidopsis, flax, soybean and many others have been successfully transformed with the nptII gene. In plants, kanamycin is the most commonly used selective agent, normally in concentrations ranging from 50 to 500 mg/l. It is very effective in inhibiting the growth of untransformed cells. However, kanamycin is ineffective as a selection marker for several legumes and gramineae. For instance, in rice, kanamycin seems to interfere with the regeneration of transformed cells to green plants. As an alternative, paromomycin can be used for selecting nptII-transformed rice cells. Therefore, the choice of the selective agent is important and based on the plant species to be transformed.
Agricultural industrial applications
Field trials and commercial releases
According to information provided by BioTrack, a database administered by the Organisation for Economic Cooperation and Development (OECD) containing records of field trials and commercial releases in OECD countries (currently 30) from 1996 to 2000, essentially all of genetically-modified organisms (GMO) are plants (98.4%). Most of the research and development of GMOs is carried out in the United States (71.1%). The rest of the OECD countries contribution to GMOs is less than 10% each, with Canada close to 9% and the other countries ranging between 5% and 0.6%. Among plants, maize is the crop with the largest number of genetically-modified varieties (37.4%) followed by oilseed rape (12.4%) and potato (12.1%).
Most introduced traits in the modified crops confer resistance to compounds such as herbicides, pests, such as insects and nematodes, and diseases caused by bacteria, fungi and viruses. Characteristics such as color of flowers, delayed ripening of fruits, and sterility have also been introduced in plants to a lesser extent. Antibiotic resistance is not a trait of interest for most of the modified plants. Nevertheless, nptII is a feature present in many plant releases because it has been used to assist in their selection.
According to the information on globally approved GM plants compiled and provided by Agriculture & Biotechnology Strategies (Canada) Inc., modified plants containing nptII gene that are approved for release into the environment as food or feed products include maize, canola (oilseed rape), melon, potato, tomato and cotton as a fiber crop. Most of the releases have occurred in the United States. However, some transformed cotton varieties developed by Monsanto have been approved in several other countries such as Australia, Argentina, Canada, China, India, Japan, Mexico and South Africa.
Multiple risk assessments of crops, including those for human consumption, containing the nptII gene and its protein have found that there are no scientific reasons to deny or restrict the use of this gene in transgenic crops on grounds of human, animal or environmental safety.