This study was funded by Canadian Institutes for Health Research (CIHR) grant. STC holds a doctoral research award from the CIHR and the Michael Smith Foundation. EYS holds a CIHR scholarship. The CIHR Transplantation Training Program also provided graduate scholarships to STC, EYS and SS.

1. L&#39;ADN de plasmide Purification <ol><li> Extraire l&#39;ADN plasmidique en utilisant un kit de maxiprep selon le protocole du fabricant. Remettre en suspension l&#39;ADN de plasmide dans 500 ul de tampon TE. </li><li> Effectuer un phénol: chloroforme: extraction de l&#39;alcool isoamylique et précipitation à l&#39;isopropanol pour éliminer les contaminants bactériens résiduels. La présence de LPS interfère avec la transfection 9. <ol><li> Ajouter 500 pi de phénol: chloroforme: alcool isoamylique (25: 24: 1, pH 8) à l&#39;ADN plasmidique et agiter vigoureusement pendant 15 sec. Phénol provoque de graves brûlures de la peau et est un anesthésique afin brûlures ne sont pas toujours senti jusqu&#39;à ce qu&#39;il y est des dommages graves. Effectuer extraction au phénol: isoamyle dans la hotte et faire preuve de prudence: chloroforme. </li></ol></li><li> Incuber le mélange pendant 5 min à température ambiante. Centrifuger à 13 000 g pendant 10 min à température ambiante. Transférer 350 ul de la phase aqueuse supérieure dans un nouveau tube de 1,5 ml de collection. </li><li> Ajouter 350 pi de tampon TE au tube contenant la phase organique inférieure. Agiter vigoureusement pendant 15 sec. Centrifuger à 13 000 g pendant 5 min à température ambiante. Transférer 350 pl de la phase aqueuse supérieure à 1,5 ml du même tube de collecte. </li><li> Ajouter 70 ul d&#39;acétate de sodium 3 M (pH 5,2) et bien mélanger. Ajouter 700 pi d&#39;isopropanol à 100%. Mélangez bien et incuber 10 min à température ambiante. Centrifugeuse à 13.000 g pendant 10 minat 4 ° C. Retirer le surnageant. </li><li> Ajouter 500 ul d&#39;éthanol à 75% au culot. échantillons de Vortex pour mélanger et centrifuger à 5000 g pendant 5 min à 4 ° C. Retirer le surnageant. ADN se trouve dans le culot. </li><li> Ouvrez le bouchon du tube et sécher à l&#39;air le culot à température ambiante pendant 5 à 10 min. Le culot doit devenir translucide. Ajouter 500 ul de tampon TE pour remettre en suspension le culot d&#39;ADN. On chauffe à 50 - 60 ° C pendant 10 minutes pour dissoudre le culot d&#39;ADN. </li><li> Mesurer l&#39;absorbance à 260 nm (A260) et 280 nm (A280) avec un spectromètre. Calculer la concentration de l&#39;ADN en multipliant la valeur de A260 par 50 ng / ul. Évaluer la qualité de l&#39;ADN par calculating le rapport A260 / A280. L&#39;ADN de haute qualité devrait donner un rapport A260 / A280 de 1,8 à 2,0. </li></ol> 2. La culture cellulaire et l&#39;ensemencement <ol><li> les cellules RAW264.7 de culture dans du DMEM supplémenté avec 9% de sérum de veau foetal (milieu DMEM / 9% de FCS) dans un 37 ° C, 5% de CO 2 incubateur. Au cours de chaque passage, détacher les cellules de la plaque par pipetage continu à l&#39;aide d&#39;une pipette Pasteur. Passage des cellules tous les 2 jours, et ensemencer 1,5 à 2.000.000 cellules sur un plat traité de culture de tissu de 10 cm que le stock. Décongeler un nouveau stock de cellules tous les 5 - 6 semaines. </li><li> Le jour de la transfection, ensemencer 200 000 cellules par puits dans une plaque à 24 puits dans un volume de 500 ul de DMEM / 9% de FCS. Incuber les cellules pendant 4 heures dans le 37 ° C incubateur. </li><li> transfecter des cellules, soit avec le réactif à base de lipides (étape 3.1) ou le réactif à base de polyamine de protéine / (étape 3.2). </li></ol> 3. La transfection <ol><li> Transfection à base de lipides: <ol><li> Réchauffer le Reagen de transfectiont à la RT dans l&#39;obscurité. Réchauffez milieu sans sérum et DMEM / 9% de FCS à 37 ° C. </li><li> Ajouter 0,5 pg d&#39;ADN de plasmide à 50 pi de milieu sans sérum dans un tube de 1,5 ml. Ajouter 2 ul de réactif de transfection avec la pointe de la pipette au-dessous de la surface du liquide. Vortex pendant 6 sec. </li><li> Laisser reposer le mélange réactif / ADN dans l&#39;obscurité à température ambiante pendant 30 min. </li><li> Au cours de l&#39;incubation de 30 min, retirez le support du puits et ajouter 250 microlitres de frais DMEM / 9% de FCS à chaque puits. </li><li> Après 30 min, ajouter 250 ul de DMEM / FCS à 9% du mélange réactif / ADN. Mélangez bien par pipetage de haut en bas. </li><li> Ajouter 300 ul du mélange dilué à chaque puits. Incuber pendant 2 à 4 h dans un 37 ° C, 5% de CO 2 incubateur. </li><li> Enlever la solution de transfection et ajouter 500 ul de DMEM / 9% de FCS. Incuber pendant 24 à 48 heures dans un 37 ° C, 5% de CO 2 incubateur avant de stimulation cellulaire. </li></ol></li><li> Protéine / transfection à base de polyamine: <ol><li> Réchauffez sans sérummoyen et DMEM / 9% de FCS à 37 ° C. </li><li> Ajouter 0,75 ul de réactif de transfection à 18,75 pi de milieu exempt de sérum. Vortex brièvement pour mélanger. Incuber à température ambiante pendant 5 min. Ajouter 0,5 pi de 1 ug / ul ADN dans le réactif transfectées diluée. Incuber à température ambiante pendant 10 min. </li><li> Pendant les 10 minutes d&#39;incubation, retirer le support de chaque puits de la plaque de 24 puits et ajouter 250 microlitres DMEM frais / 9% FCS à chaque puits. </li><li> Après 10 min, ajouter 250 pi de DMEM / FCS 9% dans le mélange réactif / ADN. </li><li> Ajouter 270 ul du mélange dilué dans le puits. Incuber dans un incubateur à CO2 à 37 ° C et 5% pour les 2 - 4 heures. </li><li> Enlever la solution de transfection et ajouter 500 ul de DMEM / 9% de FCS. Incuber pendant 24 à 48 heures dans un 37 ° C, 5% de CO 2 incubateur. </li></ol></li></ol> 4. cellulaire Stimulation et de dosage de luciférase <ol><li> Remplacez le support dans les bien avec 250 pi de DMEM frais / 9% de FCS. </li><li> Ajouter 50 ul de LPS ou LPS + IL-10 Aux concentrations désirées dans le puits. Retourner la plaque à la 37 ° C, 5% de CO 2 incubateur. Stimuler pour les 2 - 6 heures. </li><li> Enlever la solution de stimulation par aspiration, laver avec de la glace froide PBS, et ajouter 200 ul de tampon de lyse passive 1x. Rock à température ambiante pendant 30 min. Grattez et transférer le lysat tubes de 1,5 ml et éliminer les débris cellulaires par centrifugation à 20.000 g pendant 10 min à 4 ° C. </li><li> Transfert 40 pi de lysat clarifié (surnageant) à une plaque de 96 puits à fond transparent blanc pour la détermination de signaux luciférase selon le protocole du fabricant. </li><li> Lire le signal de la luciférase avec un luminomètre à travers l&#39;ensemble du spectre de la lumière visible. </li></ol> 5. Analyse des données <ol><li> Calculer la luciole: Renilla rapport en divisant les valeurs individuelles de la luciférase de luciole par les valeurs de la luciférase de Renilla dans chaque puits. </li><li> Calculer le facteur de changement en divisant la luciole: Renilla rapport de la bien traités parcelle du puits non traitée. </li></ol>

<ol>
	<li>Maurisse, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+transfection+of+DNA+into+primary+and+transformed+mammalian+cells+from+different+lineages.">Comparative transfection of DNA into primary and transformed mammalian cells from different lineages.</a> BMC Biotechnol. 10 (1), 9-18 (2010).</li><li>Thompson, C. D., Frazier-Jessen, M. R., Rawat, R., Nordan, R. P., Brown, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+methods+for+transient+transfection+of+a+murine+macrophage+cell+line+RAW+264.7.">Evaluation of methods for transient transfection of a murine macrophage cell line RAW 264.7.</a> Biotechniques. 27 (4), 824-835 (1999).</li><li>Kim, T., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Analytical and Bioanalytical Chemistry. 397 (8), 3173-3178 (2010).</li><li>Stacey, K. J., Sweet, M. J., Hume, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+ingest+and+are+activated+by+bacterial+DNA.">Macrophages ingest and are activated by bacterial DNA.</a> Journal of immunology. 157 (5), 2116-2122 (1996).</li><li>Jiang, W., Reich, I. C., Pisetsky, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+activation+of+the+RAW264.7+macrophage+cell+line+by+transfected+mammalian+DNA.">Mechanisms of activation of the RAW264.7 macrophage cell line by transfected mammalian DNA.</a> Cell Immunol. 229 (1), 31-42 (2004).</li><li>Jiang, W., Pisetsky, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+induction+of+HMGB1+release+from+RAW+264.7+cells+by+transfected+DNA.">The induction of HMGB1 release from RAW 264.7 cells by transfected DNA.</a> Mol Immunol. 45 (7), 2038-2046 (2008).</li><li>Hargreaves, D. C., Horng, T., Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+inducible+gene+expression+by+signal-dependent+transcriptional+elongation.">Control of inducible gene expression by signal-dependent transcriptional elongation.</a> Cell. 138 (1), 129-147 (2009).</li><li>Cheung, S. T., So, E. Y., Chang, D., Ming-Lum, A., Mui, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-10+inhibits+lipopolysaccharide+induced+miR-155+precursor+stability+and+maturation.">Interleukin-10 inhibits lipopolysaccharide induced miR-155 precursor stability and maturation.</a> PLoS One. 8 (8), e71336 (2013).</li><li>Weber, M., Moller, K., Welzeck, M., Schorr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+technical+reports.+Effects+of+lipopolysaccharide+on+transfection+efficiency+in+eukaryotic+cells.">Short technical reports. Effects of lipopolysaccharide on transfection efficiency in eukaryotic cells.</a> Biotechniques. 19 (6), 930-940 (1995).</li><li>Berghaus, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+responses+of+primary+murine+macrophage-lineage+cells+and+RAW+264.7+cells+to+ligands+of+Toll-like+receptors.">Innate immune responses of primary murine macrophage-lineage cells and RAW 264.7 cells to ligands of Toll-like receptors.</a> Comp Immunol Microbiol Infect Dis. 33 (5), 443-456 (2000).</li><li>Fiszer-Kierzkowska, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome-based+DNA+carriers+may+induce+cellular+stress+response+and+change+gene+expression+pattern+in+transfected+cells.">Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells.</a> BMC Mol Biol. 12 (1), 27-36 (2011).</li><li>Jacobsen, L., Calvin, S., Lobenhofer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+effects+of+transfection:+the+potential+for+misinterpretation+of+gene+expression+data+generated+from+transiently+transfected+cells.">Transcriptional effects of transfection: the potential for misinterpretation of gene expression data generated from transiently transfected cells.</a> Biotechniques. 47 (7), 617-626 (2009).</li></ol>

Les auteurs ont rien à révéler.

Le protocole décrit ici ne se concentre pas uniquement sur l&#39;efficacité de transfection, mais vise à trouver un équilibre entre l&#39;efficacité et la conservation des états physiologiques de cellules. Plus précisément, notre procédure réussit à minimiser la toxicité du réactif de transfection et de maximiser le signal luciférase. Une étape critique dans le protocole est la santé des cellules. Envahi par la végétation des cultures ne sont pas appropriés pour la transfe...

La transfection d&#39;acides nucléiques dans des cellules a une application variée dans la recherche scientifique. Des exemples comprennent (1) des gènes rapporteurs pour étudier le rôle des éléments de différents gènes dans l&#39;expression génique, (2) les plasmides d&#39;expression de protéine de surexpriment la protéine d&#39;intérêt, et (3) de petit ARN interférent régulent à la baisse l&#39;expression du gène. En manipulant le niveau de certains gènes d&#39;expression et de mesurer l&#39;effet différentiel de ces manipulations, les chercheurs peuvent déduire les fonctions des gènes dans les systèmes biologiques choisis. Toutes les méthodes de transfection fournissent les mêmes efficacités de transfection, et même le même procédé de transfection ne transfecter pas tous les types cellulaires également une. Par conséquent, différents procédés de transfection ont été développés tel que le procédé au phosphate de calcium, DEAE-dextrane, transfection lipide cationique, la transfection de polymère non-lipide cationique, une électroporation, et nucléofection 2,3. Transfection dans les macrophages est ESPEciellement difficile en raison du fait que les macrophages sont des phagocytes professionnels qui sont très sensibles à des matières étrangères, y compris les bactéries dérivées (méthyle) 4 ADN. L&#39;introduction d&#39;ADN étranger active la voie Toll-like récepteurs 9 (TLR9) conduisant à la production de cytokines et de 5,6 d&#39;oxyde nitrique. Ces macrophages activés peuvent alors être moins sensibles au traitement que les chercheurs entendent examiner. Notre laboratoire transfecte régulièrement la lignée cellulaire de macrophages RAW264.7 avec des gènes rapporteurs luciférase, et nous avons développé un protocole qui est suffisamment robuste pour activer le signal luciférase significativement plus élevé que de fond, mais aussi assez doux pour les macrophages de rester à leur état de repos. Les comportements des cellules transfectées ont été évaluées par un gène rapporteur de luciférase de luciole-porteuse de la région de promoteur de IκBζ (pGL3- IκBζ). IκBζ expression est régulée positivement par le composant lèvre de la paroi cellulaire bactérienneopolyssacharide (LPS) 7,8, et régulée à la baisse par la cytokine anti-inflammatoire, l&#39;interleukine-10 (IL-10) 8. Pour tenir compte de la variation entre les puits transfection, nous avons co-transfecter un plasmide témoin contenant le gène de la luciférase de Renilla (par exemple, phRL-TK) à des fins de normalisation. Le protocole décrit est optimisée après l&#39;essai de divers paramètres, y compris le moment de la transfection, le type de réactifs de transfection, des quantités de réactifs de transfection et de l&#39;ADN de plasmide, ainsi que le rapport de réactif de transfection de l&#39;ADN plasmidique. Les deux réactifs de transfection inclus dans ce protocole sont (1) un réactif de transfection à base de lipides et (2) une protéine / réactif de transfection à base de polyamine.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>PureLink HiPure Plasmid Maxiprep Kit</td>
			<td>Life Technologies</td>
			<td>K210007</td>
			<td>Any maxiprep kit will work</td>
		</tr>
		<tr>
			<td>Phenol:chloroform:isoamyl alcohol</td>
			<td>Life Technologies</td>
			<td>15593-049</td>
			<td>Molecular Biology Grade. Phenol is toxic so work in the fume hood, if possible. Use the lower clear organic layer if two layers of liquid form in the container.</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Scientific</td>
			<td>SH30243.01</td>
			<td>Warm in 37&#176;C water bath before use.&#160;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Scientific</td>
			<td>SH30396.03</td>
			<td>Inactivated at 56&#176;C water bath for 45 minutes before use.</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td>Warm to at least room temperature before use.</td>
		</tr>
		<tr>
			<td>XtremeGene HP DBA transfection reagent</td>
			<td>Roche</td>
			<td>6366236001</td>
			<td>Warm to room temperature before use.&#160;</td>
		</tr>
		<tr>
			<td>GeneJuice</td>
			<td>EMD Millipore</td>
			<td>70967</td>
			<td>Warm to room temperature before use.&#160;</td>
		</tr>
		<tr>
			<td>5X Passive Lysis Buffer</td>
			<td>Promega</td>
			<td>E1941</td>
			<td>30 ml is included in the Dual Luciferase Reporter Assay System</td>
		</tr>
		<tr>
			<td>Dual Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td></td>
		</tr>
	</tbody>
</table>

transfecting raw264.7 cells with a luciferase reporter gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

La figure 1 compare l&#39;efficacité de transfection des deux réactifs de transfection dans RAW264.7. Le réactif à base de lipides a typiquement taux de transfection d&#39;environ 25%, tandis que la transfection à base de polyamine de protéine / a donné lieu à environ 5% d&#39;efficacité (figure 1A). La différence dans l&#39;efficacité de transfection a été également observée dans les signaux de luciférase dans les cellules transfectées avec le RAW264.7 pGL3-promoteur I...

Watch this Scientific Journal Video about Transfecting RAW264.7 Cells with a Luciferase Reporter Gene at JoVE.com

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

Transfecter des cellules RAW264.7 avec un gène rapporteur de la luciférase

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

transfecting-raw2647-cells-with-a-luciferase-reporter-gene

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JoVE Journal

Biology

20.5K vues.  Vancouver Costal Health Research Institute. Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell’s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells. 

Vidéo: Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Transfection des cellules souches humaines à l&#39;Nucleofector Amaxa

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Recherche

Biologie

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

In vitro et in vivo en bioluminescence gène rapporteur imagerie des cellules souches embryonnaires humaines

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

Etudier biogenèse des membranes avec un Luciferase Assay-Based gène rapporteur

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

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.

Utilisation du système d&#39;électroporation Gene Pulser MXcell à transfecter des cellules primaires à haut rendement

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 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.

Aide d&#39;un compteur de cellules automatisé pour simplifier les études d&#39;expression génique: Knockdown siRNA de l&#39;IL-4 dans l&#39;expression des gènes dépendant cellules 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 ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&amp;#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

Floral-pendage de la transformation Arabidopsis thaliana Pour examiner PTSO2:: β-glucuronidase Expression du gène rapporteur

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Transfection et Nucleofecting homme cellules souches pluripotentes induites

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

L&#39;identification des cibles de microARN humaine avec le système LightSwitch Luciferase Assay utilisant 3&#39;UTR-reporter Construit et un microARN dans les cellules adhérentes Mimic

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

Surveillance de cellules autonomes Rythmes horloge circadienne de l&#39;expression génique par Reporters Bioluminescence luciférase

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Une procédure de dépistage chimique pour les glucocorticoïdes de signalisation avec un système de Reporter poisson zèbre Larve luciférase

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...