NPTII Assays for Measuring Gene Expression and Enzyme Activity in Transgenic Plants

The stable introduction of genes into plants through genetic engineering normally necessitates the use of a selectable marker, especially when the transformation frequency is low (e.g., 1.0 � 10⁻³ to 10⁻⁶ ). Marker genes enable quantification of both the transformation efficiency and gene expression (1 ). The most commonly used selectable marker is the gene from Transposon 5 (Tn5) of Escherichia coli K12 (aphA2) (2 ), which encodes aminoglycoside 3-phosphotransferase II (APH [3] II, Chemical Abstracts Registry number 58943-39-8) activity. This enzyme, also known as neomycin phosphotransferase II (NPTII), inactivates by phosphorylation the sugar-containing antibiotics, neomycin, kanamycin, geneticin (G418), and paromomycin. To date, the gene has been introduced into over 30 plant species. (For a detailed description of the nptII gene, see Chapter 1. )