Severity: Warning
Message: file_get_contents(https://...@gmail.com&api_key=61f08fa0b96a73de8c900d749fcb997acc09): Failed to open stream: HTTP request failed! HTTP/1.1 429 Too Many Requests
Filename: helpers/my_audit_helper.php
Line Number: 143
Backtrace:
File: /var/www/html/application/helpers/my_audit_helper.php
Line: 143
Function: file_get_contents
File: /var/www/html/application/helpers/my_audit_helper.php
Line: 209
Function: simplexml_load_file_from_url
File: /var/www/html/application/helpers/my_audit_helper.php
Line: 3098
Function: getPubMedXML
File: /var/www/html/application/controllers/Detail.php
Line: 574
Function: pubMedSearch_Global
File: /var/www/html/application/controllers/Detail.php
Line: 488
Function: pubMedGetRelatedKeyword
File: /var/www/html/index.php
Line: 316
Function: require_once
Severity: Warning
Message: Attempt to read property "Count" on bool
Filename: helpers/my_audit_helper.php
Line Number: 3100
Backtrace:
File: /var/www/html/application/helpers/my_audit_helper.php
Line: 3100
Function: _error_handler
File: /var/www/html/application/controllers/Detail.php
Line: 574
Function: pubMedSearch_Global
File: /var/www/html/application/controllers/Detail.php
Line: 488
Function: pubMedGetRelatedKeyword
File: /var/www/html/index.php
Line: 316
Function: require_once
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.
Download full-text PDF |
Source |
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4307275 | PMC |
http://dx.doi.org/10.3390/ijms16010788 | DOI Listing |
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