Ringraziamo il Dr. Richard Vallee e Brikha Shrestha per aiutare con il in utero ed ex tecniche di elettroporazione in vivo della retina, rispettivamente, e il dottor T. Sakurai dei commenti a questo protocollo. Questo lavoro è stato sostenuto dal National Institutes of Borse Salute F31 NS051008 (TJP), la Fondation pour la Recherche Medicale, e lo Human Frontier Science Program (AR) e R01 EY12736 (CM).

<ol>
	<li>Chalupa, L. M., Williams, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Eye Retina and the Visual Systems of the Mouse. , (2008).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proc Natl Acad Sci U S A. 101, 16-22 (2004).</li><li>Ishikawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+GDNF+gene+transfer+into+axotomized+retinal+ganglion+cells+using+in+vivo+electroporation+with+a+contact+lens-type+electrode.">Effect of GDNF gene transfer into axotomized retinal ganglion cells using in vivo electroporation with a contact lens-type electrode.</a> Gene Ther. 12, 289-298 (2005).</li><li>Donovan, S. L., Dyer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+square+wave+electroporation+of+retinal+explant+cultures.">Preparation and square wave electroporation of retinal explant cultures.</a> Nat Protoc. 1, 2710-2718 (2006).</li><li>Garcia-Frigola, C., Carreres, M. I., Vegar, C., Herrera, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+into+mouse+retinal+ganglion+cells+by+in+utero+electroporation.">Gene delivery into mouse retinal ganglion cells by in utero electroporation.</a> BMC Dev Biol. 7, 103-10 (2007).</li><li>Petros, T. J., Shrestha, B. R., Mason, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+and+sufficiency+of+EphB1+in+driving+the+ipsilateral+retinal+projection.">Specificity and sufficiency of EphB1 in driving the ipsilateral retinal projection.</a> J Neurosci. 29, 3463-3474 (2009).</li></ol>

In questo video, abbiamo dimostrato tecniche di elettroporazione, sia in utero ed ex vivo, per la consegna gene in embrioni murini RGCs. In utero elettroporazione retina fornisce un meccanismo per la manipolazione dell&#39;espressione genica in RGCs e visualizzare le loro proiezioni degli assoni in vivo. Permettendo gli embrioni permette di sopravvivere dopo la nascita esame di tali GFP + proiezioni RGC nella loro target, il nucleo genicolato laterale (LGN). Ex elettroporazione in vivo della retina fornisce una capacità più controllato a specifici target regioni della retina per l&#39;uso con saggi in vitro. Combinando in vivo e in vitro, analisi derivanti da queste tecniche permette di caratterizzazione approfondita dello sviluppo RGC e proiezioni assonale in un sottogruppo di RGCs in un contesto altrimenti normale. Mentre la nostra manifestazione si concentra sulla guida RGC assoni a livello del chiasma ottico, sia in utero ed ex electroporations vivo della retina potrebbe essere utile per studiare come perturbazioni nella differenziazione effetto espressione genica RGC, morfologia dendritiche, le proprietà elettrofisiologiche, formazione di sinapsi e propria targeting nelle zone di tale risoluzione come LGN e collicolo superiore.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>BTX Electro Square Porator ECM 830 (with foot paddle)</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>450052</td>
<td>&nbsp;</td>
<tr>
<td>Round tweezer electrodes</td>
<td>&nbsp;</td>
<td>Nepa Gene</td>
<td>CUY650-5 or CUY650-7</td>
<td>&nbsp;</td>
<tr>
<td>Silk Sutures</td>
<td>&nbsp;</td>
<td>Henry Schein</td>
<td>101-2636</td>
<td>&nbsp;</td>
<tr>
<td>Glass micropipettes with plungers</td>
<td>&nbsp;</td>
<td>Drummond</td>
<td>5-000-1001-X10</td>
<td>&nbsp;</td>
<tr>
<td>Antibiototic-antimycotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240096</td>
<td>&nbsp;</td>
<tr>
<td>DMEM/F12 medium</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11330057</td>
<td>&nbsp;</td>
<tr>
<td>Albumin bovine serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A8806-5G</td>
<td>&nbsp;</td>
<tr>
<td>ITS supplement</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I-1884</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Methyl cellulose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M0512</td>
<td>&nbsp;</td>
<tr>
<td>Glass Bottom Microwell Dishes</td>
<td>&nbsp;</td>
<td>MatTek Corporation</td>
<td>P35G-1.5-14-C</td>
<td>&nbsp;</td>
<tr>
<td>Poly-L-ornithine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4957</td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells

La retina e il suo unico neurone di output, le cellule gangliari della retina (RGC), costituiscono un ottimo modello per esaminare questioni biologiche, come la differenziazione cellulare, l&#39;orientamento degli assoni, organizzazione e formazione di sinapsi retinotopica [1]. Uno svantaggio è l&#39;impossibilità di manipolare in modo efficiente e affidabile in RGCs espressione genica in vivo, soprattutto nei percorsi murino altrimenti accessibile visiva. Topi transgenici possono essere utilizzati per manipolare l&#39;espressione genica, ma questo approccio è spesso costoso, richiede tempo, e può produrre effetti collaterali indesiderati. In pulcino, in elettroporazione ovo è usato per manipolare l&#39;espressione genica in RGCs per l&#39;esame della retina e lo sviluppo RGC. Sebbene le tecniche di elettroporazione simili sono state sviluppate in cuccioli di topo neonatale [2], ratti adulti [3], e la retina embrionale murino in vitro [4], nessuna di queste strategie permettono caratterizzazione completa dello sviluppo RGC e proiezioni degli assoni in vivo. A tal fine, abbiamo sviluppato due applicazioni di elettroporazione, una in utero e gli altri ex vivo, in maniera specifica RGCs embrionale murino [5, 6]. Con elettroporazione in utero della retina, possiamo misexpress o downregulate geni specifici in RGCs e seguire le loro proiezioni assone attraverso le vie visive in vivo, che consente l&#39;esame delle decisioni di orientamento a obiettivi intermedi, come il chiasma ottico, o in regioni-obiettivo, come il nucleo genicolato laterale. Perturbante espressione genica in un sottogruppo di RGCs in uno sfondo altrimenti wild-type facilita la comprensione della funzione genica in tutto il percorso della retina. Inoltre, abbiamo sviluppato una tecnica compagno per analizzare la crescita degli assoni RGC in vitro. Noi electroporate embrionale teste ex vivo, raccogliere e incubare la retina intero, quindi preparare espianti di retina in questi giorni più tardi. Espianti di retina può essere utilizzato in una varietà di test in vitro al fine di esaminare la risposta del elettroporate assoni RGC ai segnali di guida o altri fattori. In sintesi, questo insieme di tecniche migliora la nostra capacità di misexpress o downregulate geni RGCs e dovrebbero grande aiuto studi che hanno esaminato lo sviluppo RGC e proiezioni assone.

Watch this Scientific Journal Video about In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells at JoVE.com

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Qui vi presentiamo due tecniche per la manipolazione dell&#39;espressione genica nel topo cellule gangliari della retina (RGCs) da<em&gt; In utero</em&gt; E<em&gt; Ex vivo</em&gt; Elettroporazione. Queste tecniche permettono di esaminare come le alterazioni nell&#39;espressione genica influenzano lo sviluppo RGC, l&#39;orientamento degli assoni, e le proprietà funzionali.

In elettroporazione in vivo ed ex utero per l&#39;espressione genica in cellule gangliari della retina del mouse

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

in-utero-ex-vivo-electroporation-for-gene-expression-mouse-retinal

Research

JoVE Journal

Biology

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

18.7K Views.  Columbia University College of Physicians and Surgeons. Qui vi presentiamo due tecniche per la manipolazione dell'espressione genica nel topo cellule gangliari della retina (RGCs) da In utero E Ex vivo Elettroporazione. Queste tecniche permettono di esaminare come le alterazioni nell'espressione genica influenzano lo sviluppo RGC, l'orientamento degli assoni, e le proprietà funzionali.

Video: In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

In Utero iniezione intraventricolare e Elettroporazione di embrioni di ratto E16

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors ...

Ricerca

Biologia

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

In Utero iniezione intraventricolare e Elettroporazione di E15 embrioni di topo

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Utilizzando il sistema di elettroporazione Gene Pulser MXcell per trasfettare cellule primarie con alta efficienza

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Ricostituzione in vitro di Active T. castaneum telomerasi

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Utilizzando Sequence SECM arresto come uno strumento per isolare ribosoma Polipeptidi Bound

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

Il trasferimento genico in embrioni di pollo da anziani<em&gt; Ex ovo</em&gt; Elettroporazione

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA screening per identificare ubiquitina e ubiquitina-come regolatori del sistema di percorsi biologici in cellule di mammifero coltivate

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.