Vorremmo ringraziare Gomes E. per consigli utili e A. Wilde per averci permesso di utilizzare attrezzature varie. Questo lavoro è stato sostenuto da una borsa di AFP dal Canadian Institute for Health Research (FRN 102725).

<ol>
	<li>Luo, M. J., Reed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+is+required+for+rapid+and+efficient+mRNA+export+in+metazoans.">Splicing is required for rapid and efficient mRNA export in metazoans.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 14937-14942 (1999).</li><li>Bossie, M. A., Silver, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+of+macromolecules+between+the+cytoplasm+and+the+nucleus+in+yeast.">Movement of macromolecules between the cytoplasm and the nucleus in yeast.</a> Curr. Opin. Genet. Dev. 2, 768-774 (1992).</li><li>Farny, N. G., Hurt, J. A., Silver, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition+of+global+and+transcript-specific+mRNA+export+pathways+in+metazoans.">Definition of global and transcript-specific mRNA export pathways in metazoans.</a> Genes Dev. 22, 66-78 (2008).</li><li>Audibert, A., Weil, D., Dautry, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+kinetics+of+mRNA+splicing+and+transport+in+mammalian+cells.">In vivo kinetics of mRNA splicing and transport in mammalian cells.</a> Mol. Cell. Biol. 22, 6706-6718 (2002).</li><li>Snaar, S. P., Verdijk, P., Tanke, H. J., Dirks, 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=Kinetics+of+HCMV+immediate+early+mRNA+expression+in+stably+transfected+fibroblasts.">Kinetics of HCMV immediate early mRNA expression in stably transfected fibroblasts.</a> J. Cell. Sci. 115, 321-328 (2002).</li><li>Palazzo, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+signal+sequence+coding+region+promotes+nuclear+export+of+mRNA.">The signal sequence coding region promotes nuclear export of mRNA.</a> PLoS Biol. 5, e322-e322 (2007).</li><li>Valencia, P., Dias, A. P., Reed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+promotes+rapid+and+efficient+mRNA+export+in+mammalian+cells.">Splicing promotes rapid and efficient mRNA export in mammalian cells.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 3386-3391 (2008).</li><li>Palazzo, A. F., Eng, C. H., Schlaepfer, D. D., Marcantonio, E. E., Gundersen, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+stabilization+of+microtubules+by+integrin-+and+FAK-facilitated+Rho+signaling.">Localized stabilization of microtubules by integrin- and FAK-facilitated Rho signaling.</a> Science. 303, 836-839 (2004).</li><li>Keller, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proper+alignment+of+the+microscope.">Proper alignment of the microscope.</a> Methods Cell Biol. 72, 45-55 (2003).</li><li>Mathews, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporating+chemical+modification+constraints+into+a+dynamic+programming+algorithm+for+prediction+of+RNA+secondary+structure.">Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 7287-7292 (2004).</li><li>G&#246;rlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+signal+sequence+receptor+has+a+second+subunit+and+is+part+of+a+translocation+complex+in+the+endoplasmic+reticulum+as+probed+by+bifunctional+reagents.">The signal sequence receptor has a second subunit and is part of a translocation complex in the endoplasmic reticulum as probed by bifunctional reagents.</a> J. Cell Biol. 111, 2283-2294 (1990).</li><li>Perales, R., Bentley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Cotranscriptionality":+the+transcription+elongation+complex+as+a+nexus+for+nuclear+transactions.">"Cotranscriptionality": the transcription elongation complex as a nexus for nuclear transactions.</a> Mol. Cell. 36, 178-191 (2009).</li><li>Buratowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+through+the+RNA+polymerase+II+CTD+cycle.">Progression through the RNA polymerase II CTD cycle.</a> Mol. Cell. 36, 541-546 (2009).</li><li>Maniatis, T., Reed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extensive+network+of+coupling+among+gene+expression+machines.">An extensive network of coupling among gene expression machines.</a> Nature. 416, 499-506 (2002).</li><li>Carter, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcript+copy+number+estimation+using+a+mouse+whole-genome+oligonucleotide+microarray.">Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray.</a> Genome Biol. 6, 61-61 (2005).</li></ol>

Nessun conflitto di interessi dichiarati.

Microiniezione è un potente strumento che può essere utilizzato per studiare una serie di diversi processi cellulari. A differenza di transfezione delle cellule convenzionali, microiniezione consente al ricercatore di introdurre efficacemente gli acidi nucleici all&#39;interno dei nuclei cellulari in tempi molto stretti. Come risultato, si può eseguire analisi cinetiche come determinare il tasso di esportazione mRNA, localizzazione mRNA, e la sintesi proteica. Inoltre, poiché il periodo dell&#39;esperimento è relativamente breve, si può esporre le cellule di composti tossici in intervalli di tempo brevi, senza incorrere in effetti pleiotropici. Inoltre, composti inibitori o stimolatori, come dominante negativo proteine, può essere co-iniettata con gli acidi nucleici. Infine, microiniezioni evitare il requisito per i reagenti di trasfezione, che spesso sono tossici per le cellule e possono turbare diverse funzioni cellulari. mRNA vs iniezioni di DNA plasmidico La scelta di quale acido nucleico per iniettare dipenderà da una serie di considerazioni. Dopo l&#39;iniezione plasmide DNA, l&#39;RNA polimerasi II non solo sintetizza mRNA, ma anche fattori recluta proteine ​​per la trascrizione nascente 12,13. Dal momento che questi fattori governano gli eventi come l&#39;elaborazione di mRNA, l&#39;esportazione nucleare e di localizzazione citoplasmatica, iniezioni di DNA plasmidico può essere utilizzato per valutare come la trascrizione è accoppiata a processi a valle. Anche se la trascrizione può essere accoppiato ad esportare nucleare 14, abbiamo trovato che l&#39;mRNA iniettata viene esportato un po &#39;più veloce in vivo mRNA trascritto 6. Le ragioni di ciò non sono chiare al momento, ma questo risultato potrebbe suggerire che come l&#39;mRNA di nuova sintesi emerge dalla RNA polimerasi II, può essere legato a complessi che ritardano l&#39;esportazione nucleare. Ci sono alcuni vantaggi con iniezioni mRNA. In primo luogo, il ricercatore non deve usare gli inibitori della polimerasi RNA, che potrebbe incidere come le cellule regolano il metabolismo generale di RNA. In secondo luogo, l&#39;RNA può essere modificato in vitro prima dell&#39;iniezione. Per esempio, mRNA può essere sintetizzato con le varie 5 &#39;analoghi cappuccio o poli (A) lunghezze di coda al fine di valutare come queste caratteristiche influiscono esportazione nucleare 6. In terzo luogo, l&#39;esatta quantità di RNA che viene iniettato può essere approssimativamente stimato. Seguendo il protocollo di cui sopra, si stima che circa 20.000 a 50.000 molecole vengono iniettati in ogni nucleo, che è relativamente piccolo rispetto al numero totale di trascrizioni in una tipica cellula di mammifero (400.000 a 850.000 molecole) 15. Il principale svantaggio con iniezioni di mRNA è che durante la microiniezione, qualche perdita di liquido ad iniezione nel citoplasma è inevitabile. Dal momento che l&#39;mRNA iniettato direttamente nel citoplasma è stabile per lunghi periodi (A. Palazzo, osservazione inedito), particolare attenzione deve essere presa per selezionare quali cellule sono quantificati. Generalmente si analizzano le cellule che hanno ricevuto&gt; 90% del fluido di iniezione nel nucleo, valutato mediante la distribuzione di OG-destrano.

<table><tbody><tr><td>Injection buffer</td><td>Sigma-Aldrich</td><td></td><td>140 mM KCl and 10 mM HEPES, pH 7.4</td></tr><tr><td>Oregon Green 488-70kD Dextran</td><td>Invitrogen</td><td>D7173</td><td>Stock of 10mg/ml in injection buffer can be stored for longs periods of time at -80&amp;#x00B0;C</td></tr><tr><td>Borosilicate capillary tubes</td><td>World Precision Instruments, Inc.</td><td>1B100F-4</td><td></td></tr><tr><td>Gel loading pipette tips for loading injection needles</td><td>Eppendorf</td><td>022351656</td><td></td></tr><tr><td>Microscope</td><td>Nikon Instruments</td><td>Eclipse Ti-S</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige International</td><td>NT-88-V3MSH</td><td></td></tr><tr><td>Syringe for injection</td><td>BD Biosciences</td><td>512311</td><td></td></tr><tr><td>PBS Buffer</td><td>Sigma-Aldrich</td><td></td><td>137 mM NaCl, 2.7 mM KCl, 10 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 2 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, pH 7.4</td></tr><tr><td>SSC Buffer</td><td>Sigma-Aldrich</td><td></td><td>150 mM NaCl, 15 mM sodium citrate, pH 7.4</td></tr><tr><td>4% Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15686</td><td>Stock of 37% Paraformaldehyde is diluted in PBS</td></tr><tr><td>0.1% Triton X-100 (Surfact-Amps)</td><td>Thermo Fisher Scientific, Inc.</td><td>28314</td><td>Stock of 10% Triton X-100 is diluted in PBS</td></tr><tr><td>Formamide</td><td>Fisher Scientific</td><td>F84-1</td><td></td></tr><tr><td>Dextran sulfate</td><td>Fisher Scientific</td><td>BP1585-100</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; tRNA</td><td>Sigma-Aldrich</td><td>R1753</td><td></td></tr><tr><td>Vanadyl riboside complex (VRC)</td><td>Sigma-Aldrich</td><td>R3380</td><td></td></tr><tr><td>Whatman filter paper</td><td>VWR international</td><td>28298-020</td><td></td></tr><tr><td>Vibration control table</td><td>Kinetic Systems</td><td>5702E-3036-21</td><td></td></tr><tr><td>Fluoromount G</td><td>SouthernBiotech</td><td>0100-20</td><td></td></tr><tr><td>&amp;alpha;-amanitin</td><td>Sigma-Aldrich</td><td>A2263</td><td></td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>13-374-12</td><td></td></tr><tr><td>Flaming/Brown Micropipette Puller</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>Vacuum Grease</td><td>Dow Corning</td><td>695400008</td><td></td></tr><tr><td>T7 RNA Polymerase</td><td>New England Biolabs</td><td>M0251S</td><td></td></tr><tr><td>Poly(A) Polymerase</td><td>Ambion</td><td>AM1350</td><td></td></tr><tr><td>Hybridization Buffer</td><td>Sigma-Aldrich</td><td></td><td>100mg/ml dextran sulfate, 5mM VRC, 150mM NaCl, 15mM sodium citrate, 0.5mg/ml &lt;em&gt;E. coli&lt;/em&gt; tRNA, 60% formamide</td></tr></tbody></table>

analysis of mrna nuclear export kinetics in mammalian cells by microinjection

Negli eucarioti, RNA messaggero (mRNA) è trascritto nel nucleo e devono essere esportati nel citoplasma per accedere al macchinario di traduzione. Anche se l&#39;esportazione nucleare di mRNA è stata ampiamente studiata in ovociti di Xenopus 1 e gli organismi geneticamente docile come lievito 2 e la linea cellulare derivata Drosophila S2 3, pochi studi erano stati condotti in cellule di mammifero. Inoltre, la cinetica di esportazione mRNA in cellule somatiche dei mammiferi poteva solo essere dedotta indirettamente 4,5. Per misurare la cinetica esportazione nucleare di mRNA nelle cellule di mammifero coltura tissutale, abbiamo sviluppato un metodo che utilizza la potenza di microiniezione accoppiata con ibridazione in situ fluorescente (FISH). Questi test sono stati utilizzati per dimostrare che in cellule di mammifero, la maggior parte degli mRNA vengono esportati in modo splicing dipendente 6,7, o in modo che richiede specifiche sequenze di RNA come la regione sequenza segnale di codifica (SSCR) 6. In questo saggio, le cellule sono microiniettati sia con mRNA sintetizzato in vitro o DNA plasmide contenente il gene di interesse. Le cellule vengono incubate per microiniettati vari momenti poi fissa e la localizzazione sub-cellulare di RNA è valutata mediante FISH. A differenza di transfezione, dove la trascrizione avviene alcune ore dopo l&#39;aggiunta di acidi nucleici, la microiniezione di DNA o mRNA permette di espressione rapida e consente la generazione di precisi dati cinetici.

Watch this Scientific Journal Video about Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection at JoVE.com

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

Qui si descrive un metodo che utilizza la potenza di microiniezione accoppiato con fluorescente<em&gt; In situ</emIbridazione&gt; al fine di misurare con precisione la cinetica esportazione nucleare di mRNA nelle cellule somatiche dei mammiferi.

Analisi della cinetica di esportazione mRNA nucleare in cellule di mammifero da Microiniezione

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

15.2K Views.  University of Toronto. Qui si descrive un metodo che utilizza la potenza di microiniezione accoppiato con fluorescente In situ al fine di misurare con precisione la cinetica esportazione nucleare di mRNA nelle cellule somatiche dei mammiferi.

Video: Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

L&#39;espressione di proteine ​​fluorescenti in<em&gt; Branchiostoma lanceolatum</em&gt; By mRNA iniezione in ovociti non fecondate

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Ricerca

Biologia dello sviluppo

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Usando i Oocytes del Mouse per valutare la funzione del Gene umano durante la meiosi I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Genetica

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microiniezione degli ovociti di Xenopus Laevis

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Biologia

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

Un saggio reporter per analizzare la maturazione dei microRNA intronici in cellule di mammifero

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves ...

Ricerca sul cancro

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Microiniezione intranucleari di DNA in dissociato neuroni dei mammiferi adulti

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Analisi per misurare il trasporto nucleocitoplasmatico in tempo reale all&#39;interno di motori neuronici come le cellule NSC-34

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Neuroscienze

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

Metodo per l&#39;isolamento e l&#39;identificazione di mRNA, microRNA e componenti proteiche dei complessi Ribonucleoproteina da estratti cellulari con RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

Visualizzazione di mRNA reticolo endoplasmatico localizzate in cellule di mammifero

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

Analisi della localizzazione snRNA Spliceosomic nelle cellule umane di Hela utilizzando la microiniezione

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Analisi della cinetica di esportazione mRNA nucleare in cellule di mammifero da Microiniezione

Riepilogo

Esplora Altri Video

Capitoli in questo video

L'espressione di proteine fluorescenti in

Usando i Oocytes del Mouse per valutare la funzione del Gene umano durante la meiosi I

Microiniezione degli ovociti di Xenopus Laevis

Un saggio reporter per analizzare la maturazione dei microRNA intronici in cellule di mammifero

Microiniezione intranucleari di DNA in dissociato neuroni dei mammiferi adulti

Analisi per misurare il trasporto nucleocitoplasmatico in tempo reale all'interno di motori neuronici come le cellule NSC-34

Metodo per l'isolamento e l'identificazione di mRNA, microRNA e componenti proteiche dei complessi Ribonucleoproteina da estratti cellulari con RIP-Chip

Visualizzazione di mRNA reticolo endoplasmatico localizzate in cellule di mammifero

Analisi della localizzazione snRNA Spliceosomic nelle cellule umane di Hela utilizzando la microiniezione

Meiotic mandrino valutazione in mouse Ovociti mettendo a tacere siRNA-mediata

Analisi della cinetica di esportazione mRNA nucleare in cellule di mammifero da Microiniezione

Riepilogo

Esplora Altri Video

Capitoli in questo video

L'espressione di proteine ​​fluorescenti in

Usando i Oocytes del Mouse per valutare la funzione del Gene umano durante la meiosi I

Microiniezione degli ovociti di Xenopus Laevis

Un saggio reporter per analizzare la maturazione dei microRNA intronici in cellule di mammifero

Microiniezione intranucleari di DNA in dissociato neuroni dei mammiferi adulti

Analisi per misurare il trasporto nucleocitoplasmatico in tempo reale all'interno di motori neuronici come le cellule NSC-34

Metodo per l'isolamento e l'identificazione di mRNA, microRNA e componenti proteiche dei complessi Ribonucleoproteina da estratti cellulari con RIP-Chip

Visualizzazione di mRNA reticolo endoplasmatico localizzate in cellule di mammifero

Analisi della localizzazione snRNA Spliceosomic nelle cellule umane di Hela utilizzando la microiniezione

Meiotic mandrino valutazione in mouse Ovociti mettendo a tacere siRNA-mediata

L'espressione di proteine fluorescenti in