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:[add a comment]
- 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.[add a comment]
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.[add a comment]
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.[add a comment]
The information contained in this page was believed to be correct at the time it was collated. New patents and patent applications, altered status of patents, and case law may have resulted in changes in the landscape. CAMBIA makes no warranty that it is correct or up to date at this time and accepts no liability for any use that might be made of it. Corrections or updates to the information are welcome. Please send an email to firstname.lastname@example.org.