Name: analysis of mrna nuclear export kinetics in mammalian cells by microinjection
Uploaded: 2010-12-04T13:00:00
Duration: 11 min 32 s
Description: Watch this Scientific Journal Video about Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection at JoVE.com

We would like to thank E. Gomes for useful advice and A. Wilde for allowing us to use various equipment. This work was supported by a grant to A.F.P. from the 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>

No conflicts of interest declared.

Microinjection is a powerful tool that can be used to study a number of different cellular processes. Unlike conventional cell transfection, microinjection allows the researcher to effectively introduce nucleic acids into the cellular nuclei within a very narrow timeframe. As a result, one can perform kinetic analyses such as determining the rates of mRNA export, mRNA localization, and protein synthesis. Furthermore, since the timeframe of the experiment is relatively brief, one can expose cells to toxic compounds within short time spans without incurring pleiotropic effects. Moreover, inhibitory or stimulatory compounds such as dominant negative proteins, can be co-injected with the nucleic acids. Finally, microinjections avoid the requirement for transfection reagents, which are often toxic to cells and may perturb different cellular functions.
mRNA vs. plasmid DNA injections
The choice of which nucleic acid to inject will depend on a number of considerations. Following plasmid DNA injection, RNA Polymerase II not only synthesizes mRNA, but also recruits protein factors to the nascent transcript12,13. Since these factors govern events such as mRNA processing, nuclear export and cytoplasmic localization, plasmid DNA injections can be used to assess how transcription is coupled to downstream processes. Although transcription may be coupled to nuclear export14, we have found that injected mRNA is exported slightly faster than in vivo transcribed mRNA6. The reasons for this are not clear at the moment, but this result may suggest that as newly synthesized mRNA emerges from RNA Polymerase II, it may be tied up in complexes that retard nuclear export.
There are some advantages with mRNA injections. First, the researcher does not have to use RNA Polymerase inhibitors, which might affect how cells regulate the overall metabolism of RNA. Secondly, the RNA can be modified in vitro prior to injection. For instance, mRNAs can be synthesized with various 5' cap analogues or poly(A) tail lengths in order to assess how these features affect nuclear export6. Thirdly, the exact amount of RNA that is injected can be roughly estimated. Following the protocol outlined above, we estimate that approximately 20,000 to 50,000 molecules are injected in each nuclei, which is relatively small when compared to the total number of transcripts in a typical mammalian cell (400,000 to 850,000 molecules)15. The major disadvantage with mRNA injections is that during microinjection, some leakage of injection fluid into the cytoplasm is unavoidable. Since mRNA injected directly into the cytoplasm is stable over long periods (A. Palazzo, unpublished observation), extra care must be taken to select which cells are quantified. Generally we analyze cells that received &gt;90% of the injection fluid in the nucleus, as assessed by the distribution of OG-dextran.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>Injection buffer</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>&nbsp;</td>
<td>140 mM KCl and 10 mM HEPES, pH 7.4</td>
</tr>
<tr>
<td>Oregon Green 488-70kD Dextran</td>
<td>&nbsp;</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&deg;C</td>
</tr>
<tr>
<td>Borosilicate capillary tubes</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>1B100F-4</td>
<td>&nbsp;</td>
<tr>
<td>Gel loading pipette tips for loading injection needles</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>022351656</td>
<td>&nbsp;</td>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>Eclipse Ti-S</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>Narishige Group</td>
<td>NT-88-V3MSH</td>
<td>&nbsp;</td>
<tr>
<td>Syringe for injection</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>512311</td>
<td>&nbsp;</td>
<tr>
<td>PBS Buffer</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>&nbsp;</td>
<td>137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4</td>
</tr>
<tr>
<td>SSC Buffer</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>&nbsp;</td>
<td>150 mM NaCl, 15 mM sodium citrate, pH 7.4</td>
</tr>
<tr>
<td>4% Paraformaldehyde</td>
<td>&nbsp;</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>&nbsp;</td>
<td>Thermo Scientific</td>
<td>28314</td>
<td>Stock of 10% Triton X-100 is diluted in PBS</td>
</tr>
<tr>
<td>Formamide</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>F84-1</td>
<td>&nbsp;</td>
<tr>
<td>Dextran sulfate</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>BP1585-100</td>
<td>&nbsp;</td>
<tr>
<td>E. coli tRNA</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>R1753</td>
<td>&nbsp;</td>
<tr>
<td>Vanadyl riboside complex (VRC)</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>R3380</td>
<td>&nbsp;</td>
<tr>
<td>Whatman filter paper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>28298-020</td>
<td>&nbsp;</td>
<tr>
<td>Vibration control table</td>
<td>&nbsp;</td>
<td>Kinetic Systems</td>
<td>5702E-3036-21</td>
<td>&nbsp;</td>
<tr>
<td>Fluoromount G</td>
<td>&nbsp;</td>
<td>Southern Biotech</td>
<td>0100-20</td>
<td>&nbsp;</td>
<tr>
<td>&alpha;-amanitin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>A2263</td>
<td>&nbsp;</td>
<tr>
<td>Parafilm</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>13-374-12</td>
<td>&nbsp;</td>
<tr>
<td>Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument Co.</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum Grease</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>695400008</td>
<td>&nbsp;</td>
<tr>
<td>T7 RNA Polymerase</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>M0251S</td>
<td>&nbsp;</td>
<tr>
<td>Poly(A) Polymerase</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1350</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization Buffer</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>&nbsp;</td>
<td>100mg/ml dextran sulfate, 5mM VRC, 150mM NaCl, 15mM sodium citrate, 0.5mg/ml E. coli tRNA, 60% formamide</td>
</tr>		</tbody>
		</table>
		</div>

analysis of mrna nuclear export kinetics in mammalian cells by microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

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

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

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

analysis-mrna-nuclear-export-kinetics-mammalian-cells

Research

JoVE Journal

Biology

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

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.

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

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...