लेजर Microdissection से कोशिकाओं के सबसेट के जीन अभिव्यक्ति रूपरेखा लागू करने के लिए ड्रोसोफिला विंग डिस्क
Summary
लेजर microdissection ड्रोसोफिला विंग डिस्क के विशिष्ट डिब्बों में जीन अभिव्यक्ति की रूपरेखा का विश्लेषण स्थानीयकृत आरएनएआई के अधीन करने के लिए लागू किया गया था<em> Vivo में</em>. खामोश और unsilenced डिब्बों के बराबर क्षेत्रों से निकाले आरएनए मात्रात्मक RT-पीसीआर द्वारा विश्लेषण किया गया था देशी ऊतक microecology के संदर्भ में तुलनात्मक जीन अभिव्यक्ति की रूपरेखा निर्धारित करने के लिए.
Abstract
Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut.
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.
Protocol
Discussion
अपने बेसल अभिव्यक्ति स्तर unsilenced पंख डिस्क के पूर्वकाल / पीछे डिब्बों में हासिल करने के लिए सम्मान के साथ, चयनित जीन एक्स की गतिविधि मुंह बंद करने पर 40% तक कम पाया गया. इसके विपरीत, अपने ख्यात लक्ष्य (जीन वाई) …
Acknowledgements
लेखकों मुनाफे धन्यवाद. Chiara Campanella और क्षमता के AMRA केंद्र, विश्वविद्यालय नेपल्स Federico द्वितीय के, नेपल्स, इटली लेजर microdissector के उपयोग के साथ उन्हें प्रदान करने के लिए, और 3 डी एनीमेशन में उदार मदद के लिए श्री Vincenzo Vicidomini.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| DEPC water | Sigma | W4502 | ||
| RNase Zap | Sigma | R2020 | ||
| TRI Reagent | Sigma | T9424 | ||
| SuperScript III | Invitrogen | 18080093 | ||
| Master Mix | Invitrogen | 11761100 | ||
| Agarose | Sigma | A9539 | ||
| Isopropyl alcohol | Sigma | I9516 | ||
| Chloroform | Sigma | C7559 | ||
| Dream taq | Fermentas | EP0701 |
Fly strains
Drosophila UAS-silencing lines can be obtained from VDRC RNAi collection 2. A collection of GAL4-driver lines is available at the Bloomington Drosophila Stock Center (Indiana, USA).
Equipment
Required equipment includes Laser microdissector (Leica LMD6000), Coulter Microfuge 22R (Beckman), PCR (BIO-RAD My Cycler), Real Time PCR (BIO-RAD iQ5).
Tools
Required tools include: glass wells, brush, microdissection forceps, hanging drop slides, insect pins mounted on syringe needles, metal frames with PET membrane, plastic tubes (50 ml, 05 ml, 0.25 ml).
References
- Elliott, D. A., Brand, A. H., Dahmann, C. The GAL4 System A Versatile System for the Expression of Gene. , (2008).
- Klein, T. Wing disc development in the fly: the early stages. . Current Opinion in Genetics & Development. 11, 470-475 (2001).
- Dietzl, G. A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature. 448, 151-U1-151-U1 (2007).
- Martin, F. A., Morata, G. Compartments and the control of growth in the Drosophila wing imaginal disc. Development. 133, 4421-4426 (2006).
- Tabata, T. Genetics of morphogen gradients. Nature Reviews Genetics. 2, 620-630 (2001).
- Moreno, E. &. a. m. p. ;. a. m. p., Basler, K. dMyc transforms cells into super-competitors. Cell. 117, 117-129 (2004).
