प्रतिदीप्ति सक्रिय संयंत्र Protoplasts के सेल छंटनी
Summary
संयंत्र सामग्री के विशिष्ट प्रकार की कोशिकाओं से अलग करने के लिए एक विधि का प्रदर्शन है. इस तकनीक को विशेष प्रकार की कोशिकाओं, सेलुलर हदबंदी और प्रतिदीप्ति सक्रिय सेल छंटनी में ट्रांसजेनिक मार्कर फ्लोरोसेंट प्रोटीन व्यक्त लाइनों कार्यरत हैं. इसके अतिरिक्त, एक विकास सेटअप यहाँ की स्थापना की है कि के उपचार की सुविधा<em> Arabidopsis thaliana</em> सेल छँटाई पूर्व seedlings.
Abstract
High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.
Protocol
Discussion
Protoplasts, सिद्धांत रूप में, कर सकते हैं संयंत्र के ऊतकों की एक किस्म से प्राप्त किया जा, अनुकूल परिस्थितियों के अनुकूलन बहुत शाही सेना गुणवत्ता और मात्रा में वृद्धि होगी. दोनों protoplasting समाधान और वैकल्पिक ऊष्म…
Acknowledgements
यह काम राष्ट्रीय विज्ञान फाउंडेशन (अनुदान नहीं 0519984 DBI) और राष्ट्रीय स्वास्थ्य संस्थान के (अनुदान नहीं 5R01GM078279.) द्वारा समर्थित किया गया था ..
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| 250 μm nylon mesh | Sefar Filtration | NITEX 03-250/50 | ||
| 100 μm nylon mesh | Sefar Filtration | NITEX 03-100/47 | ||
| Square petri dishes | Fisher Scientific | 08-757-10k | ||
| Phytatrays | Sigma | P1552 | ||
| Murashige and Skoog Basal Medium (MS) | Sigma | M5519 | ||
| sucrose | Fisher Scientific | S5-3 | ||
| MES | Sigma | M2933 | ||
| KOH | Sigma | P1767 | 10 M stock | |
| Eclipse 90i microscope | Nikon | |||
| Cellulase R-10 | Yakult Pharmaceutical | |||
| Macerozyme R-10 | Yakult Pharmaceutical | |||
| D-mannitol | Sigma | M9546 | ||
| KCl | Sigma | P8041 | 1 M stock | |
| BSA | Sigma | A3912 | ||
| β-mercaptoethanol | CALBIOCHEM | 444203 | ||
| CaCl2 | Sigma | C2536 | 1 M stock | |
| orbital shaker | LAB-LINE | |||
| 40 μm cell strainer | BD Falcon | 352340 | ||
| conical 15 ml tubes | BD Falcon | 352196 | ||
| table centrifuge | Sorvall | Legend RT | ||
| NaCl | Sigma | S3014 | ||
| FACSAria | BD | |||
| 1.5 ml microfuge tubes | VWR | 20170-38 | ||
| RNeasy micro kit | QIAGEN | 74004 | ||
| WT-Ovation Pico RNA Amplification System | NuGEN | 3300_12 | ||
| FL-Ovation cDNA Biotin Module V2 | NuGEN | 4200_12 |
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