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Int. J. Mol. Sci. 2015, 16(1), 788-804; doi:10.3390/ijms16010788

Improvement of the Fluorescence Intensity during a Flow Cytometric Analysis for Rice Protoplasts by Localization of a Green Fluorescent Protein into Chloroplasts

1
National Academy of Agricultural Science, Rural Development Administration, Jeonju 560-500, Korea
2
Graduate School of Biotechnology and Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea
3
Gyeonggi Bio-Center, Gyeonggi Institute of Science & Technology Promotion, Suwon 443-270, Korea
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Academic Editor: Hatem Rouached
Received: 29 August 2014 / Accepted: 22 December 2014 / Published: 31 December 2014
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Abstract

Protoplasts have been a useful unicellular system for various molecular biological analyses based on transient expression and single cell analysis using fluorescence-activated cell sorting (FACS), widely used as a powerful method in functional genomics. Despite the versatility of these methods, some limits based on low fluorescence intensity of a flow cytometric analysis (FCA) using protoplasts have been reported. In this study, the chloroplast targeting of fluorescent proteins (FPs) led to an eight-fold increase in fluorescence intensity and a 4.5-fold increase of transfection ratio from 14.7% to 65.7% as compared with their targeting into the cytoplasm. Moreover, the plot data of FCA shows that 83.3% of the K-sGFP population is under the threshold level, regarded as a non-transgenic population with background signals, while 65.7% of the K-sGFP population is spread on overall intervals. To investigate the reason underlying this finding, mRNA/protein levels and transfection efficiency were analyzed, and results suggest that mRNA/protein levels and transfection ratio are not much different between K-sGFP and KR-sGFP. From those results, we hypothesized that the difference of fluorescence intensity is not only derived from cellular events such as molecular level or transfection efficiency. Taken together, we suggest that the translocation of FPs into chloroplasts contributes to the improvement of fluorescence intensity in FCA and, apparently, plays an important role in minimizing the loss of the transfected population. Our study could be usefully applicable for highly sensitive FACS and FCA-investigations of green tissue. View Full-Text
Keywords: chloroplast; flow cytometric analysis (FCA); protoplast; rice; transit peptide; fluorescence-activated cell sorting (FACS); synthetic green fluorescent protein (sGFP) chloroplast; flow cytometric analysis (FCA); protoplast; rice; transit peptide; fluorescence-activated cell sorting (FACS); synthetic green fluorescent protein (sGFP)
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

You, M.K.; Lim, S.-H.; Kim, M.-J.; Jeong, Y.S.; Lee, M.-G.; Ha, S.-H. Improvement of the Fluorescence Intensity during a Flow Cytometric Analysis for Rice Protoplasts by Localization of a Green Fluorescent Protein into Chloroplasts. Int. J. Mol. Sci. 2015, 16, 788-804.

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