A sensitive and simple paper chromatographic procedure for detecting neomycin phosphotransferase II (NPTII) gene expression

1990 ◽  
Vol 14 (5) ◽  
pp. 873-876 ◽  
Author(s):  
Pranab Roy ◽  
Nirmala Sahasradbudhe
Keyword(s):  
Gene Expression ◽  
Nptii Gene ◽  
Neomycin Phosphotransferase ◽  
Neomycin Phosphotransferase Ii ◽  
Chromatographic Procedure

A rapid immunoprecipitation assay for neomycin phosphotransferase II expression in transformed bacteria and plant tissues

1990 ◽  
Vol 68 (6) ◽  
pp. 983-987
Author(s):  
Chris L. Baszczynski
Keyword(s):  
Brassica Napus ◽  
Bovine Serum Albumin ◽  
Kanamycin Resistance ◽  
Plant Tissues ◽  
Nptii Gene ◽  
Neomycin Phosphotransferase ◽  
Kanamycin Resistance Gene ◽  
Dot Blot ◽  
Immunoprecipitation Assay ◽  
Neomycin Phosphotransferase Ii

Anti-kanamycin antibodies produced in rabbits, following coupling of the antibiotic to bovine serum albumin, were used to immunoprecipitate radioactively labelled phosphorylated kanamycin from transformed bacterial or plant extracts in a novel assay system, for the detection of neomycin phosphotransferase II (NPTII) activity. Radioactive counts in the immunoprecipitated pellet give a semiquantitative measure of the kanamycin phosphorylation and hence the amount of NPTII activity. This assay is sensitive, uses very small amounts of radioactivity, and is very rapid, allowing many samples to be processed within a few hours. Immunoprecipitated counts from reactions with bacteria carrying a kanamycin resistance gene or from tobacco and Brassica napus plants transformed with NPTII gene-containing vectors were consistently higher than counts from nontransformed controls. Results obtained with this assay correlate well with those from the previously described gel overlay and dot-blot assays, but can be obtained in an appreciably shorter time frame.Key words: anti-kanamycin antibodies, immunoprecipitation, neomycin phosphotransferase II assay, transformation.

Microprojectile-mediated DNA delivery to the Salicaceae family

1993 ◽  
Vol 71 (11) ◽  
pp. 1458-1466 ◽  
Author(s):  
Yvonne A. Devantier ◽  
Barbara Moffatt ◽  
Catherine Jones ◽  
Pierre J. Charest
Keyword(s):  
Gene Expression ◽  
Populus Deltoides ◽  
Expression System ◽  
Transient Gene Expression ◽  
Dna Delivery ◽  
Kanamycin Resistance ◽  
Cell Suspensions ◽  
Neomycin Phosphotransferase ◽  
Salix Alba ◽  
Neomycin Phosphotransferase Ii

A transient gene expression system was developed for the Salicaceae family using microprojectile-mediated DNA delivery (Biolistic™) to cell suspensions. Using Populus nigra × Populus maximowiczii line NM1, 10 variables were optimized. Optimum transient gene expression under the 10 conditions tested was obtained with a 7- to 9-day-old cell suspension. Highest transient levels were observed with samples positioned 13.5 cm from the stopping plate and bombarded with 1.6-μm gold particles coated with 1 μg of DNA precipitated with CaCl2. Assaying for β-glucuronidase gene expression was performed 1 day after bombardment. The fate of the microparticles in the bombarded cells was studied, showing that 58.9% of cells expressing the β-glucuronidase gene had microparticles located in the nucleus or its vicinity and the remaining cells had microparticles in the cytoplasm. Cell suspensions from five different lines (P. nigra × P. maximowiczii lines NM1 and NM6, Populus deltoides × P. nigra line DN106, Populus tremula × Populus alba line 7171-B4, and Salix alba sanguined line SA-2) yielded transient gene expression. The relative strengths of β-glucuronidase expression in the lines tested were NM1 = 7171-B4 > NM6 > DN106 > SA-2. Six plasmid constructions were also tested in line NM1 for transient β-glucuronidase gene expression. The x-glucuronide histochemical assay did not reveal any differences, but the methyl umbelliferone glucuronide fluorescent assay yielded the following relative levels of transient gene expression with the different promoter sequences: 35S-35S-AMV enhancer = 35S-AMV enhancer > 35S-35S = 35S-35S-AMV enhancer with the β-glucuronidase – neomycin phosphotransferase fusion > 35S. Four transgenic cell lines of P. nigra × P. maximowiczii were characterized for kanamycin resistance and neomycin phosphotransferase II gene activity. Polymerase chain reaction and Southern hybridization analyses indicated the presence of the β-glucuronidase and neomycin phosphotransferase II genes in the genome of three of these transgenic lines. Key words: microprojectile, particle bombardment, Salicaceae, poplar, willow.

scholarly journals Successful genetic transformation in date palm (Phoenix dactylifera)

2014 ◽  
Vol 11 (2) ◽  
pp. 171-176 ◽  
Author(s):  
L Hassan
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Agrobacterium Rhizogenes ◽  
Reporter Gene ◽  
Hairy Roots ◽  
Date Palm ◽  
Binary Vector ◽  
Nptii Gene ◽  
Neomycin Phosphotransferase ◽  
Gus Gene ◽  
Nos Terminator

The introduction of foreign genes into most of the Phoenix spp using recombinant DNA technology is not a straight forward task. In Phoenix spp application of this technology towards successful transformation proved to be a more difficult one – so far no report on the successful regeneration of transgenic date palm plants has been published. We developed an efficient and reproducible variety-independent method for producing transgenic date palm (Phoenix spp) via Agrobacterium-mediated transformation. Agrobacterium rhizogenes strains LBA 9402 were used and for cotransformation experiments the strain LBA 9402 with the binary vector pBIN19 with the p35S GUS INT gene was used. Off-shoot segments from different Phoenix spp cultivars were infected with Agrobacterium rhizogenes. The development of ‘hairy roots’ at a high frequency only on infected tissue pieces showed that transformation is possible. Various parameters like, effect of different genotypes on root initiation, root number and root length have been studied. Regeneration of transformed root cultures to plantlets was also attempted. Histochemical GUS assay and polymerase chain reaction analysis of hairy roots confirmed the presence of GUS gene. Agrobacterium tumifaciensmediated transformation was also performed using the leaves of off-shoot explants. Agrobacterium tumefaciens strains: I) GV3101 with the vir plasmid pMP90 the strain C58C1 ATHV with the vir-plasmid pTiBo542 (=pEHA101; Hood et al. 1986) was used. The nptII gene (neomycin phosphotransferase) was used as a selectable marker gene. The ?-Glucuronidase-gene (GUS-Gene: Jefferson et al. 1987) under control of the Ubi- and 35S-Promotors, with an Intron (Vancanneyt et al. 1990), was used as the reporter gene. We also used the genetically engineered Agrobacterium tumefaciens strain LBA4404 as a vector for infection in the transformation experiment, which contains plasmid pBI121 of 14 KDa (binary vector). This binary vector contains following genes within the right border (RB) and left border (LB) region of the construct: The udiA gene (Jefferson, 1986) predetermining GUS (?-glucuronidase), driven by CaMV promoter and NOS terminator. This reporter gene can be used to assess the efficiency of transformation. The nptII gene (Herrera-Estrella et al., 1983) encoding neomycin phosphotransferase II (nptII) conferring kanamycin resistance, driven by NOS promoter and NOS terminator. The bacterium also contains plasmid pAL4404 which is a disarmed Ti plasmid (132 KDa) containing the virulence genes. For the confirmation of transgenes, calli were taken from the growing callus mass for DNA isolation. PCR- and Southern analysis was performed to determine the integration and the copy number of the transgene. The GUS-test was performed to demonstrate ß-glucuronidase expression. The transgenic plantlets were kept in a hardening room for four weeks and they will be transferred to a growth chamber with controlled environment for further establishment. DOI: http://dx.doi.org/10.3329/jbau.v11i2.19841 J. Bangladesh Agril. Univ. 11(2): 171-176, 2013

scholarly journals Use of the gene encoding neomycin phosphotransferase II to convect linked markers into the vaccinia virus genome

1988 ◽  
Vol 16 (4) ◽  
pp. 1634-1634 ◽  
Author(s):  
Christine A. Franke ◽  
Dennis E. Hruby
Keyword(s):  
Vaccinia Virus ◽  
Virus Genome ◽  
Neomycin Phosphotransferase ◽  
Gene Encoding ◽  
Neomycin Phosphotransferase Ii

Fusion proteins with both insecticidal and neomycin phosphotransferase II activity

FEBS Letters ◽  
1988 ◽  
Vol 226 (2) ◽  
pp. 364-370 ◽  
Author(s):  
Herman Höfte ◽  
Saskia Buyssens ◽  
Mark Vaeck ◽  
Jan Leemans
Keyword(s):  
Fusion Proteins ◽  
Neomycin Phosphotransferase ◽  
Neomycin Phosphotransferase Ii

Evaluation of transgenic canola plants under field conditions

Genome ◽  
1992 ◽  
Vol 35 (1) ◽  
pp. 58-63 ◽  
Author(s):  
M. Arnoldo ◽  
C. L. Baszczynski ◽  
G. Bellemare ◽  
G. Brown ◽  
J. Carlson ◽  
...  
Keyword(s):  
Kanamycin Resistance ◽  
Genetically Engineered ◽  
Field Conditions ◽  
Enzyme Assays ◽  
Nptii Gene ◽  
Neomycin Phosphotransferase ◽  
Agrobacterium Mediated Transformation ◽  
Transgenic Canola ◽  
Progeny Analysis ◽  
Transformation Vectors

Eleven independent transgenic canola (Brassica napus ssp. oleifera L. cv. Westar and Regent) lines were evaluated in the field. The plants carried a neomycin phosphotransferase (NPTII) gene for kanamycin resistance that was introduced via Agrobacterium-mediated transformation. NPTII enzyme assays, Southern blot hybridizations and progeny analysis, confirmed the stable, heritable integration and expression of the introduced NPTII gene. A number of agronomic characteristics evaluated under field conditions, including maturity, yield, and oil and protein content, were all statistically comparable between the transformed and nontransformed plants. These results indicate that canola can be genetically engineered successfully, and that the Agrobacterium-based transformation system employed does not induce any adverse effects on the intrinsic agronomic and qualitative traits critical to the agricultural industry.Key words: transgenic field trial, canola, Agrobacterium-mediated transformation, vectors.

A simplified method for the detection of neomycin phosphotransferase II activity in transformed plant tissues

1987 ◽  
Vol 5 (4) ◽  
pp. 380-386 ◽  
Author(s):  
Raymond E. McDonnell ◽  
Robert D. Clark ◽  
Wendy A. Smith ◽  
Maud A. Hinchee
Keyword(s):  
Plant Tissues ◽  
Neomycin Phosphotransferase ◽  
Neomycin Phosphotransferase Ii ◽  
Simplified Method
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