 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 01, 2009 |
| Title |
Positive fluorescent selection permits precise, rapid and in-depth over-expression analysis in plant protoplasts |
| Organism |
Arabidopsis thaliana |
| Experiment type |
Expression profiling by array
|
| Summary |
Transient genetic modification of plant protoplasts is a straightforward and rapid technique for the analysis of numerous aspects of plant biology. One drawback in the analysis of transformed protoplast suspensions is that they are a heterogeneous mix of cells that have and have not been successfully transfected. To overcome this problem, we have developed a system that employs a fluorescent positive selection marker in combination with flow cytometric analysis as well as fluorescence activated cell sorting (FACS) to isolate responses in the transfected protoplasts exclusively. This recombinase-compatible system enables high-throughput screening of genetic circuitry. Moreover, the use of FACS allows in depth downstream analysis. Lastly, over-expression is an effective means to dissect regulatory networks, especially where redundancy exists. Here, this system has been applied to the study of auxin signaling in order to investigate reporter gene activation and genome-wide transcriptional changes in response to manipulation of the auxin-response network. We have transiently over-expressed dominant negative mutant isoforms of Aux/IAA transcription factors (IAA7mII and IAA19mII; Tiwari et al., 2001) in Arabidopsis Pwer::GFP root protoplasts, making use of a RFP fluorescent positive selection marker and FACS to isolate the dually labeled (IAAnmII expressing and Pwer::GFP-positive) cells. We have compared the transcriptional differences between an empty vector control, IAA7mII and IAA19mII protoplasts that had either been treated with 5microM IAA or mock-treated for 3 hours.
Keywords: hormone treatment, genetic modification
|
| |
|
| Overall design |
18 samples with 3 replicates for each condition and transformation vector: 3x empty vector mock treated, 3x empty vector IAA treated, 3x IAA7mII over-expressor mock treated, 3x IAA7mII over-expressor IAA treated, 3x IAA19mII over-expressor mock treated and 3x IAA19mII over-expressor IAA treated.
|
| |
|
| Contributor(s) |
Birnbaum KD, Bargmann BO |
| Citation(s) |
19168642 |
| |
| Submission date |
Dec 01, 2008 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Kenneth David Birnbaum |
| E-mail(s) |
[email protected]
|
| Phone |
212-998-8257
|
| Organization name |
New York University
|
| Department |
Biology
|
| Lab |
Birnbaum
|
| Street address |
12 Waverly Place
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10003 |
| Country |
USA |
| |
|
| Platforms (1) |
| GPL198 |
[ATH1-121501] Affymetrix Arabidopsis ATH1 Genome Array |
|
| Samples (18)
|
| GSM346829 |
empty vector_mock treated_replicate 1 |
| GSM346830 |
empty vector_mock treated_replicate 2 |
| GSM346831 |
empty vector_mock treated_replicate 3 |
| GSM346832 |
empty vector_IAA treated_replicate 1 |
| GSM346833 |
empty vector_IAA treated_replicate 2 |
| GSM346834 |
empty vector_IAA treated_replicate 3 |
| GSM346835 |
IAA7mII over-expressor_mock treated_replicate 1 |
| GSM346836 |
IAA7mII over-expressor_mock treated_replicate 2 |
| GSM346837 |
IAA7mII over-expressor_mock treated_replicate 3 |
| GSM346838 |
IAA7mII over-expressor_IAA treated_replicate 1 |
| GSM346839 |
IAA7mII over-expressor_IAA treated_replicate 2 |
| GSM346840 |
IAA7mII over-expressor_IAA treated_replicate 3 |
| GSM346841 |
IAA19mII over-expressor_mock treated_replicate 1 |
| GSM346842 |
IAA19mII over-expressor_mock treated_replicate 2 |
| GSM346843 |
IAA19mII over-expressor_mock treated_replicate 3 |
| GSM346844 |
IAA19mII over-expressor_IAA treated_replicate 1 |
| GSM346845 |
IAA19mII over-expressor_IAA treated_replicate 2 |
| GSM346846 |
IAA19mII over-expressor_IAA treated_replicate 3 |
|
| Relations |
| Affiliated with |
GSE69995 |
| BioProject |
PRJNA110613 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE13783_RAW.tar |
33.6 Mb |
(http)(custom) |
TAR (of CEL, EXP) |
| Processed data included within Sample table |
|
|
|
|
 |
Less...
More...