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. Plasmid DNA Purification <ol><li> Extrahieren von Plasmid-DNA unter Verwendung eines Maxiprep-Kit nach Vorschrift des Herstellers. Resuspendieren Plasmid-DNA in 500 ul TE Puffer. </li><li> Führen eine Phenol: Chloroform: Isoamylalkohol-Extraktion und Isopropanol-Fällung Rest bakterielle Verunreinigungen zu entfernen. Die Anwesenheit von LPS stört Transfektion 9. <ol><li> Gib 500 ul Phenol: Chloroform: Isoamylalkohol (25: 24: 1, pH 8) mit der Plasmid-DNA und kräftig geschüttelt, für 15 sec. Phenol verursacht schwere Verätzungen der Haut und ist ein Narkosemittel so Verbrennungen sind nicht immer das Gefühl, bis es zu schweren Schäden. Führen Phenol: Chloroform: Isoamylalkohol-Extraktion in der Abzugshaube und seien Sie vorsichtig. </li></ol></li><li> Inkubieren der Mischung für 5 min bei RT. Zentrifugieren bei 13.000 xg für 10 min bei RT. Übertragen Sie 350 ul der obere wässrige Phase in ein neues 1,5-ml-Collection-Tube. </li><li> Mit 350 ul TE-Puffer zu dem Rohr, das die untere organische Phase enthält. Kräftig schütteln für 15 Sekunden. Zentrifugieren bei 13.000 xg für 5 Minuten bei RT. Übertragen Sie 350 ul der oberen wässrigen Phase auf den gleichen 1,5-ml-Collection-Tube. </li><li> In 70 ul 3 M Natriumacetat (pH 5,2) und gut mischen. Add 700 ul 100% iges Isopropanol. Gut mischen und inkubieren 10 min bei RT. Zentrifuge bei 13.000 g für 10 MiNaT 4ºC. Entfernen Sie den Überstand. </li><li> Gib 500 &amp; mgr; l 75% Ethanol zu dem Pellet. Wirbel Proben bei 5000 xg mischen und Zentrifuge 5 min bei 4 ° C. Entfernen Sie den Überstand. DNA in dem Pellet. </li><li> Öffnen Sie den Deckel des Röhrchens und der Luft trocknen das Pellet bei RT 5 - 10 min. Das Pellet sollte durchscheinend. Gib 500 ul TE-Puffer, um das DNA-Pellet zu resuspendieren. Erwärmen bei 50 - 60 ° C für 10 min, um das DNA-Pellet zu lösen. </li><li> Die Absorption bei 260 nm (A260) und 280 nm (A280) mit einem Spektrometer. Berechnen Sie die Konzentration von DNA durch Multiplikation der A260-Wert von 50 ng / &amp; mgr; l. Bewerten Sie die Qualität der DNA durch calculating die A260 / A280-Verhältnis. 2.0 - DNA mit hoher Qualität sollte eine A260 / A280-Verhältnis von 1,8 zu ergeben. </li></ol> 2. Zellkultur und Seeding <ol><li> Kultur RAW264.7-Zellen in DMEM, ergänzt mit 9% fötales Kälberserum (DMEM / 9% FCS) in einem 37 ° C, 5% CO 2 -Inkubator. Bei jedem Durchgang, nehmen die Zellen von der Platte durch kontinuierliches Pipettieren mit einer Pasteurpipette. Passage die Zellen alle 2 Tage, und Samen von 1,5 bis 2.000.000 Zellen auf einer 10 cm Gewebekulturschale behandelt als Lager. Tauwetter ein neues Lager von Zellen alle 5 - 6 wochen. </li><li> Am Tag der Transfektion wurden Samen 200.000 Zellen pro Vertiefung in einer Platte mit 24 Vertiefungen in einem Volumen von 500 &amp; mgr; l DMEM / 9% FCS. Inkubieren der Zellen für 4 Stunden in den 37 ° C-Inkubator. </li><li> Transfektion von Zellen, die entweder mit dem Lipid-Reagens (Schritt 3.1) oder dem Protein / Polyamin-Reagens (Schritt 3.2). </li></ol> 3. Transfektion <ol><li> Lipid basierende Transfektion: <ol><li> Aufwärmen der Transfektion reagent, um in der Dunkelheit auf RT. Warm up Serum-freien Medium und DMEM / 9% FCS auf 37 ° C. </li><li> Mit 0,5 ug Plasmid-DNA in 50 ul serumfreiem Medium in einem 1,5-ml-Röhrchen. Add 2 ul Transfektionsreagenz mit der Pipettenspitze unterhalb der Oberfläche der Flüssigkeit. Vortex 6 Sek. </li><li> Verlassen das Reagenz / DNA-Mischung im Dunkeln bei RT für 30 min. </li><li> Während der 30-minütigen Inkubation, entfernen Sie die Medien aus dem Brunnen und fügen Sie 250 ul frischem DMEM / 9% FCS in jede Vertiefung. </li><li> Nach 30 min, fügen 250 ul DMEM / 9% FCS zu dem Reagenz / DNA-Gemisch. Gut mischen durch Auf-und Abpipettieren. </li><li> Werden 300 ul des verdünnten Gemisches in jede Vertiefung. Inkubieren für 2-4 h in einem 37 ° C, 5% CO 2 -Inkubator. </li><li> Entfernen Transfektionslösung und fügen Sie 500 ul DMEM / 9% FCS. Inkubieren für 24 bis 48 h in einem 37 ° C, 5% CO 2 -Inkubator vor der Zellstimulation. </li></ol></li><li> Protein / Polyamin basierende Transfektion: <ol><li> Warm up Serum-freienmittlere und DMEM / 9% FCS auf 37 ° C. </li><li> Hinzuzufügen 0,75 ul Transfektionsreagenz auf 18,75 &amp; mgr; l serumfreies Medium. Vortex kurz mischen. Inkubieren bei Raumtemperatur für 5 min. Mit 0,5 ul 1 ug / ul DNA in die verdünnte Reagenz transfiziert. Inkubieren bei Raumtemperatur für 10 min. </li><li> Während der 10-minütigen Inkubation, entfernen Medien aus jeder Vertiefung der 24-Well-Platte und fügen Sie 250 ul frischem DMEM / 9% FCS in jede Vertiefung. </li><li> Nach 10 min, fügen 250 ul DMEM / 9% FCS in das Reagenz / DNA-Gemisch. </li><li> Add 270 ul der verdünnten Mischung auf das gut. Inkubieren bei 37 ° C und 5% CO 2 Inkubator für 2-4 Stunden. </li><li> Entfernen Transfektionslösung und fügen Sie 500 ul DMEM / 9% FCS. Inkubieren für 24 bis 48 h in einem 37 ° C, 5% CO 2 -Inkubator. </li></ol></li></ol> 4. Zellstimulation und Luciferase Assay <ol><li> Tauschen Sie Medien in den gut 250 ul frischem DMEM / 9% FCS. </li><li> In 50 ul LPS oder LPS + IL-10 In den gewünschten Konzentrationen zu der Bohrung. Gibt die Platte an die 37 ° C, 5% CO 2 Inkubator. Regen Sie für 2 - 6 h. </li><li> Entfernen Stimulationslösung durch Absaugen, Waschen mit eiskaltem PBS, und fügen Sie 200 ul 1x Passive Lysis Buffer. Rock bei RT für 30 min. Kratzen und übertragen das Lysat auf 1,5-ml-Röhrchen und entfernen Zelltrümmer durch Zentrifugation bei 20.000 × g für 10 min bei 4 ° C. </li><li> Transfer 40 ul geklärtes Lysat (Überstand), um einen weißen, clear bottom Platte mit 96 Vertiefungen für die Bestimmung der Luciferase-Signalen gemäß dem Protokoll des Herstellers. </li><li> Lesen Sie das Luciferase-Signal mit einem Luminometer über das gesamte sichtbare Lichtspektrum. </li></ol> 5. Datenanalyse <ol><li> Berechnung der Firefly Renilla Verhältnis durch Dividieren einzelnen Werte von Firefly Luziferase durch die Werte von Renilla-Luciferase aus jeder Vertiefung. </li><li> Berechnen Sie die fache Änderung durch Division der Glühwürmchen: Renilla Verhältnis der behandelten gutder unbehandelten gut. </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>

Die Autoren haben nichts zu offenbaren.

Das hier beschriebene Protokoll nicht allein auf die Transfektionseffizienz zu konzentrieren, sondern zielt darauf ab, einen Ausgleich zwischen Effizienz und Erhaltung der physiologischen Zustände der Zellen zu schlagen. Insbesondere gelingt es unserem Verfahren bei der Minimierung der Toxizität von Transfektionsreagenz und Maximierung der Luciferase-Signal. Ein kritischer Schritt im Protokoll ist die Gesundheit der Zellen. Wuchert Kulturen nicht geeignet sind für die Transfektion als ihr...

Transfektion von Nukleinsäuren in Zellen hat eine vielfältige Anwendung in der wissenschaftlichen Forschung. Beispiele umfassen (1) Reportergene, die Rolle des anderen Gen Elemente Genexpression zu untersuchen, (2) Protein-Expressionsplasmiden, um das Protein von Interesse überexprimiert, und (3) kleine interferierende RNA die Genexpression herunterregulieren. Durch Manipulation des Expressionsniveaus bestimmter Gene und Messen des Differentialwirkung solcher Manipulationen können Forscher die Genfunktionen in den ausgewählten biologischen Systemen abzuleiten. Nicht alle Transfektionsverfahren die gleichen Transfektionseffizienzen und sogar die gleichen Transfektionsverfahren nicht alle Zelltypen ebenso 1 transfizieren. Daher verschiedene Transfektionsverfahren entwickelt worden, wie beispielsweise Calciumphosphat-Verfahren, DEAE-Dextran, kationische Lipid-Transfektion, kationische Lipid-Polymer nicht-Transfektion, Elektroporation und Nukleofektion 2,3. Transfektion in Makrophagen ist ESPEsondere schwierig aufgrund der Tatsache, dass Makrophagen sind professionelle Phagozyten, die sehr empfindlich gegenüber Fremdmaterialien einschließlich Bakterien stammt (methyliert) DNA 4 sind. Einführung fremder DNA aktiviert den Toll-like Rezeptor 9 (TLR9) Weg führt zu der Produktion von Cytokinen und Stickstoffmonoxid 5,6. Diese aktivierten Makrophagen können dann weniger, die auf die Behandlung, die die Forscher beabsichtigen, zu untersuchen. Unser Labor routinemäßig transfiziert den RAW264.7 Makrophagen-Zelllinie mit Luciferase-Reporter-Gene, und wir haben ein Protokoll, das robust genug ist, um die Luciferase-Signal deutlich höher als Hintergrund zu haben ist auch sanft genug für Makrophagen an ihren Ruhezustand bleiben entwickelt, aber. Die Verhaltensweisen der transfizierten Zellen wurden durch ein Glühwürmchen-Luciferase-Reporter-Gen beherbergen, das die Promotorregion des IκBζ (pGL3- IκBζ) bewertet. IκBζ Expression durch die Bakterienzellwand-Komponente Lippe hochreguliertopolyssacharide (LPS) 7,8 und durch die anti-inflammatorische Cytokin regulierte, Interleukin-10 (IL-10) 8. Um für die Transfektion Variation unter Brunnen Konto, kooperieren wir Transfektion ein Steuer Plasmid, das das Renilla-Luciferase-Gen (zB phRL-TK) zur Normalisierung Zwecke. Das beschriebene Protokoll wird nach der Prüfung verschiedener Parameter einschließlich Timing Transfektion Typ Transfektionsreagenzien Mengen von Transfektionsreagenzien und von Plasmid-DNA sowie Verhältnis von Transfektionsreagenz Plasmid-DNA optimiert. Die beiden Transfektionsreagenzien in diesem Protokoll enthalten sind (1) eine auf Lipid basierenden Transfektionsreagenz, und (2) ein Protein / Polyaminbasis Transfektionsreagenz.

<table><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 &amp;deg;C water bath before use.</td></tr><tr><td>Fetal bovine serum</td><td>Thermo Scientific</td><td>SH30396.03</td><td>Inactivated at 56 &amp;deg;C water bath for 45 min 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.</td></tr><tr><td>GeneJuice</td><td>EMD Millipore</td><td>70967</td><td>Warm to room temperature before use.</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.

Figur 1 vergleicht die Transfektionseffizienz der beiden Transfektionsreagenzien in RAW264.7. Das Lipid-Reagens ergab typischerweise etwa 25% Transfektionsrate während das Protein / Polyamin-basierende Transfektion führte zu etwa 5% Wirkungsgrad (1A). Der Unterschied in der Transfektionseffizienz wurde auch in Luciferase Signale in RAW264.7-Zellen mit den pGL3-Promotorreporter IκBζ (1B) transfiziert beobachtet. Zugabe von LPS zu diesen transfizierten Zellen erhöhte...

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.

Transfizieren RAW264.7-Zellen mit einem Luciferase-Reportergen

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

Research

JoVE Journal

Biology

20.6K Aufrufe.  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. 

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

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

In vivo Dual-Substrat Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Forschung

Medizin

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical challenges, it remains difficult to assess the activity of this signaling pathway and interpret it within a biological context. The existing literature on large tumor suppressor (LATS) relies on methods that are qualitative and cannot easily be scaled-up for screening. Recently, we have developed a bioluminescence-based biosensor to monitor the kinase activity of LATS-a core component of the Hippo kinase cascade. Here, we describe procedures for how this LATS biosensor (LATS-BS) can be used to characterize Hippo pathway regulators. First, we provide a detailed protocol for investigating the effect of an overexpressed protein candidate (e.g., VEGFR2) on LATS activity using the LATS-BS. Then, we show how the LATS-BS can be used for a small-scale kinase inhibitor screen. This protocol can feasibly be scaled-up to perform larger screens, which undoubtedly will identify novel regulators of the Hippo pathway.

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor

Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

Überwachung von Hippo Signalisierung Pathway-Aktivität unter Verwendung eines Biosensors Luciferase-basierte großen Tumorsuppressor (LATS)

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical ...

Krebsforschung

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.

Studieren Membrane Biogenesis mit einem 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 ...

Biologie

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Identifizierung von Transkriptionsfaktor-Regulierern mit Medium-Throughput-Screening von Arrayed Libraries und einem Dual-Luciferase-basierten Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

Überwachung des Brustkrebswachstums und der metastasierenden Koloniebildung bei Mäusen mittels Biolumineszenz

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

Biolumineszenz-Bildgebung von einem immunkompetenten Tiermodell für Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

In vitro Transkription und Capping von Gaussia-Luciferase mRNA von HeLa-Zelle Transfektion Gefolgt

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

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.

Überwachung Zell-autonome circadiane Uhr Rhythms der Genexpression mittels Luciferase Biolumineszenz Reporter

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

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Hochdurchsatz-Screening mit einem Funktionshomemade Dual-Luciferase Assay-Schein

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.