12 April 2005 Spectroscopic detection of fluorescent protein marker gene activity in genetically modified plants
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Proceedings Volume 5852, Third International Conference on Experimental Mechanics and Third Conference of the Asian Committee on Experimental Mechanics; (2005) https://doi.org/10.1117/12.621891
Event: Third International Conference on Experimental Mechanics and Third Conference of the Asian Committee on Experimental Mechanics, 2004, -, Singapore
Abstract
This work focuses on developing a portable fibre optic fluorescence analyser for rapid identification of genetically modified plants tagged with a fluorescent marker gene. Independent transgenic tobacco plant lines expressing the enhanced green fluorescence protein (EGFP) gene were regenerated following Agrobacterium-mediated gene transfer. Molecular characterisation of these plant lines was carried out at the DNA level by PCR screening to confirm their transgenic status. Conventional transgene expression analysis was then carried out at the RNA level by RT-PCR and at the protein level by Western blotting using anti-GFP rabbit antiserum. The amount of plant-expressed EGFP on a Western blot was quantified against known amounts of purified EGFP by scanning densitometry. The expression level of EGFP in transformed plants was found to range from 0.1 - 0.6% of total extractable protein. A comparison between conventional western analysis of transformants and direct spectroscopic quantification using the fibre optic fluorescence analyser was made. The results showed that spectroscopic measurements of fluorescence emission from strong EGFP expressors correlated positively with Western blot data. However, the fluorescence analyser was also able to identify weakly expressing plant transformants below the detection limit of colorimetric Western blotting.
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O. W. Liew, Jenny P. C. Chong, Anand K. Asundi, "Spectroscopic detection of fluorescent protein marker gene activity in genetically modified plants", Proc. SPIE 5852, Third International Conference on Experimental Mechanics and Third Conference of the Asian Committee on Experimental Mechanics, (12 April 2005); doi: 10.1117/12.621891; https://doi.org/10.1117/12.621891
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