Neomycin Phosphotransferase Gene, Kanamycin, Transgenic Plants, Detoxifies, Phosphorylation, Radioactively Labelled

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Gene Transfer Methods in Plants Gene Transfer Methods in Plants Introduction Target Cells for Transformation Theoretical Routes for Production of Transgenic Plants Vectors of Gene Transfer Vectors based on Ti and Ri Plasmids of Agrobacterium Structure and Function of Ti and Ri Plasmids Selectable Marker Genes in Vectors Used for Gene Transfer T-DNA Transfer Process Vectors based on Ti and Ri Plasmids Virus Vectors Transformation Techniques Using Agrobacterium Prerequisites for Production of Transgenic Plants Explants Used for Transformation Marker Genes for Selection and Scoring of Tranformed Cells/Calli or Shoots Reporter Genes Used as Scoreable Markers and their Enzyme Assays Neomycin Phosphotransferase Gene (nptII) Chloramphenicol Acetyl Transferase Gene (cat)

Home >> Plant Biotechnology and Genomics >> Gene Transfer Methods in Plants >>Neomycin Phosphotransferase Gene (nptll).

Neomycin Phosphotransferase Gene (nptll).
This gene is used both as a selectable and a scoreable marker in experiments involving transfer of genes leading to the production of transgenic plants. It imparts kanamycin resistance, so that the transformed tissue can be selected on kanamycin. An assay for NPTII enzyme is also used to detect its presence in transformed tissue or transgenic plants. The gene for NPTII enzyme is often used with nos promoter, which drives its synthesis. In some cases, nptll gene had adverse effect on theexpression of the desirable gene introduced (eg. bt2 gene for insect resistance; see next chapter), so that alternative approaches for improving itsexpression had to be used.

For an enzyme assay of NPTII, the enzyme is first fractionated using non-denaturing polyacrylamide gel electrophoresis (PAGE). Since the enzyme detoxifies kanamycin by phosphorylation, radioactively labelled ATP (p³²) is used with kanamycin in an agar layer, which is used' to cover the gel containing the enzyme. The whole set is incubated at 35°C and the phosphorylation leading to incorporation of ³²p in kanamycin can be detected by autoradiography. As indicated in, a more convenient and easier enzyme assay can be conducted using dot blots of cell extracts (no DNA or enzyme extraction is necessary) obtained on a filter. The filter with dot blots is incubated with the substrate (kanamycin) and is then subjected to autoradiography to detect the presence of NPTII enzyme.

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β-Glucuronidase (GUS)Gene (uidA) Luciferase Gene (lux) Green Fluorescent Protein Gene Phosphomannose Isomerase Gene (pmi) Agrobacterium-Mediated Transformation in Monocots Agroinfection and Gene Transfer Physical Delivery or DNA Mediated Gene Transfer (DMGT) Chemically Stimulated DNA Uptake by Protoplasts Microinjection and Macroinjection Microprojectiles for Gene Transfer Electroporation Method for Gene Transfer Liposome Mediated Gene Transfer Calcium Phosphate Precipitation Method Transformation Using Pollen or Pollen Tube Incubation of Dry Seeds,Embryos,Tissues or Cell in DNA Transformation by Ultrasonication Removal of Selectable Marker Gene from Transgenic Plants Need for Removal of Selectable Marker Gene Genetic Systems Used for Removal of Marker Gene Examples of North American Plant Transformation Facilities Plant Transformation Facilities (PTFs)