Name: expression analysis of mammalian linker-histone subtypes
Uploaded: 2012-03-19T12:00:00
Description: Watch this Scientific Journal Video about Expression Analysis of Mammalian Linker-histone Subtypes at JoVE.com

עבודה זו נתמכת על ידי NIH מענק GM085261 ואת הקואליציה סרטן גאורגיה פרס נכבדים Scholar (כדי YF).

1. לדוגמא הכנת והפקת RNA <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לפני מיצוי RNA, משטחים את כל העבודה במידה טפטפות יש לנגב עם אתנול 70% וטופלו עם פתרון טיהור RNase, כגון RNase זאפ. תרגול פעולה זו מפחיתה את הסיכוי לזיהום RNase והשפלה RNA. ללבוש כפפות על כל הנהלים. </li><li style=";text-al...

<ol>
	<li>Fan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H1+linker+histones+are+essential+for+mouse+development+and+affect+nucleosome+spacing+in+vivo.">H1 linker histones are essential for mouse development and affect nucleosome spacing in vivo.</a> Mol. Cell Biol. 23, 4559-4572 (2003).</li><li>Woodcock, C. L., Skoultchi, A. I., Fan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+linker+histone+in+chromatin+structure+and+function:+H1+stoichiometry+and+nucleosome+repeat+length.">Role of linker histone in chromatin structure and function: H1 stoichiometry and nucleosome repeat length.</a> Chromosome Res. 14, 17-25 (2006).</li><li>Shen, X., Gorovsky, M. 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U.S.A. 100, 5920-5925 (2003).</li><li>Happel, N., Doenecke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H1+and+its+isoforms:+contribution+to+chromatin+structure+and+function.">Histone H1 and its isoforms: contribution to chromatin structure and function.</a> Gene. 431, 1-12 (2009).</li><li>Clausell, J., Happel, N., Hale, T. K., Doenecke, D., Beato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H1+subtypes+differentially+modulate+chromatin+condensation+without+preventing+ATP-dependent+remodeling+by+SWI&#47;SNF+or+NURF.">Histone H1 subtypes differentially modulate chromatin condensation without preventing ATP-dependent remodeling by SWI&#47;SNF or NURF.</a> PLoS One. 4, 0007243-0007243 (2009).</li><li>Khadake, J. R., Rao, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-+chromatin-condensing+properties+of+rat+testes+H1a+and+H1t+compared+to+those+of+rat+liver+H1bdec;+H1t+is+a+poor+condenser+of+chromatin.">DNA- chromatin-condensing properties of rat testes H1a and H1t compared to those of rat liver H1bdec; H1t is a poor condenser of chromatin.</a> Biochemistry. 34, 15792-15801 (1995).</li><li>Orrego, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+affinity+of+mammalian+histone+H1+somatic+subtypes+for+DNA+and+chromatin.">Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin.</a> BMC Biol. 5, 22-22 (2007).</li><li>Th'ng, J. P., Sung, R., Ye, M., Hendzel, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H1+family+histones+in+the+nucleus.+Control+of+binding+and+localization+by+the+C-terminal+domain.">H1 family histones in the nucleus. Control of binding and localization by the C-terminal domain.</a> J. Biol. Chem. 280, 27809-27814 (2005).</li><li>Wang, Z. F., Sirotkin, A. M., Buchold, G. M., Skoultchi, A. I., Marzluff, W. 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+mouse+histone+H1+genes:+gene+organization+and+differential+regulation.">The mouse histone H1 genes: gene organization and differential regulation.</a> J. Mol. Biol. 271, 124-138 (1997).</li><li>Brown, D. T., Sittman, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+through+overexpression+and+tagging+of+the+variant+type+of+the+mouse+H1e+and+H1c+genes.">Identification through overexpression and tagging of the variant type of the mouse H1e and H1c genes.</a> J. Biol. Chem. 268, 713-718 (1993).</li><li>Fan, Y., Sirotkin, A., Russell, R. G., Ayala, J., Skoultchi, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+somatic+H1+subtypes+are+dispensable+for+mouse+development+even+in+mice+lacking+the+H1(0)+replacement+subtype.">Individual somatic H1 subtypes are dispensable for mouse development even in mice lacking the H1(0) replacement subtype.</a> Mol. Cell. Biol. 21, 7933-7943 (2001).</li><li>Fan, Y., Skoultchi, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+H1+linker+histone+subtypes+and+their+functions+in+mice.">Genetic analysis of H1 linker histone subtypes and their functions in mice.</a> Methods Enzymol. 377, 85-107 (2004).</li><li>Heid, C. A., Stevens, J., Livak, K. J., Williams, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+quantitative+PCR.">Real time quantitative PCR.</a> Genome Res. 6, 986-994 (1996).</li></ol>

קבוצה של מבחני שהוצג לאפשר ניתוח מקיף של רמות הביטוי של יונקים תת היסטון מקשר. מתוכנן כראוי qRT-PCR מבחני לספק מדידות רגישות גבוהה ומדויקת של הודעות רנ&quot;א של כל הגנים יונקים היסטון H1. חלק חשוב של qRT-PCR מבחני עבור מקשר גנים תת היסטון הוא הכנת cDNA באמצעות צבע יסוד על בסיס אקר...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td></tr><tr><td> RNase זאפ </td><td> יישומי Biosystems </td><td> AM9780 </td></tr><tr><td> Trizol מגיב </td><td> Invitrogen </td><td> 15596-018 </td></tr><tr><td> SuperScriptIII </td><td> Invitrogen </td><td> 18080-051 </td></tr><tr><td> Absolute אתנול </td><td> פישר סיינטיפיק </td><td> BP2818-4 </td></tr><tr><td> IQ SYBR ירוק </td><td> ביו רד </td><td> 170-8880 </td></tr><tr><td> ערכת RNeasy מיני </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> אני Deoxyribonuclease </td><td> סיגמא </td><td> המגבר-D1 </td></tr><tr><td> Microseal 96-PCR גם צלחת </td><td> ביו רד </td><td> MSP-9605 </td></tr><tr><td> אטמים דבק &#39;ב&#39; Microseal </td><td> ביו רד </td><td> MSB-1001 </tד&gt; </tr><tr><td> סוכרוז </td><td> Agros Organics </td><td> AC40594 </td></tr><tr><td> פוספט נתרן dibasic heptahydrate (Na 2 HPO 4 · 7H 2 O) </td><td> פישר סיינטיפיק </td><td> BP332 </td></tr><tr><td> נתרן כלורי (NaCl) </td><td> האמריקאי Bioanalytical </td><td> AB01915 </td></tr><tr><td> נתרן dihydrogen heptahydrate פוספט (לאא 2 PO 4 · 7H2O) </td><td> פישר סיינטיפיק </td><td> BP-330 </td></tr><tr><td> HEPES </td><td> פישר סיינטיפיק </td><td> BP310 </td></tr><tr><td> מעכב השלם מיני proteinase קוקטייל Tablet </td><td> רוש ומדע יישומי </td><td> 11836153001 </td></tr><tr><td> EDTA </td><td> סיגמא </td><td> E-5134 </td></tr><tr><td> Phenylmethanesulfonyl פלואוריד (PMSF) </td><td> האמריקאי Bioanalytical </td><td> AB01620 </td></tr><tr><td> Nonidet-40 (NP-40) </td&gt; <td> האמריקאי Bioanalytical </td><td> AB01425 </td></tr><tr><td> אשלגן כלורי (KCl) </td><td> פישר סיינטיפיק </td><td> BP366 </td></tr><tr><td> טריס [hydroxymethyl aminomethane] </td><td> האמריקאי Bioanalytical </td><td> AB02000 </td></tr><tr><td> מגנזיום כלוריד (MgCl2) </td><td> פישר סיינטיפיק </td><td> BP214 </td></tr><tr><td> חומצה גופרתית (H2SO4) </td><td> VWR </td><td> VW3648-3 </td></tr><tr><td> אמוניום הידרוקסיד (NH 4 OH) </td><td> Agros Organics </td><td> AC42330 </td></tr><tr><td> ברדפורד Assay חלבון </td><td> ביו רד </td><td> 500-0001 </td></tr><tr><td> אצטוניטריל </td><td> EMD </td><td> AX0145-1 </td></tr><tr><td> Trifluoroacetic חומצה (TFA) </td><td> JTBaker </td><td> 9470-01 </td></tr></tbody></table>

expression analysis of mammalian linker-histone subtypes

היסטון H1 מקשר נקשר החלקיקים ליבת הנוקלאוזום ו-DNA מקשר, בהנחיית מתקפל של הכרומטין למבנה מסדר גבוה יותר. H1 הוא חיוני להתפתחות היונקים 1 ומווסת ביטוי גנים ספציפיים in vivo 2-4. בין חלבונים היסטון השמורים באבולוציה, משפחת ההיסטונים H1 מקשר הוא קבוצה הטרוגנית ביותר. ישנם 11 תת H1 ביונקים אשר מוסדר באופן דיפרנציאלי במהלך הפיתוח בסוגי תאים שונים. אלה כוללים תת H1 5 סומטי H1s (H1a אלקטרוני), H1 החלפת 0, 4 תאים נבט תת ספציפיים H1, ואת H1x 5. נוכחות של מספר רב של תת H1 שונים בזיקה DNA מחייב יכולת הדחיסה הכרומטין 6-9 מספק רמה נוספת של אפנון של תפקוד הכרומטין. לכן, ניתוח ביטוי כמותי של תת H1 בודדים, הן של ה-mRNA וחלבונים, יש צורך להבין טוב יותר את הרגולציה של גבוההכרומטין מנת מבנה ותפקוד. כאן אנו מתארים סדרה של מבחני המיועדים לניתוח רמות הביטוי של תת H1 בודדים (איור 1). ביטוי mRNA של גנים שונים H1 חלופה נמדדת על ידי מערכת של רגישות גבוהה וכמותית שעתוק-PCR (qRT-PCR) מבחני הפוכה, אשר מהיר יותר, מדויק יותר ודורשים דגימות הרבה פחות לעומת הגישה החלופית של ניתוח כתם הצפון. שלא כמו רוב הודעות ה-mRNA אחרים הסלולר, mRNAs עבור גנים היסטון ביותר, כולל רוב הגנים H1, חסר זנב ארוך פולה, אבל מכילים מבנה גזע לולאה על האזור מתורגמת &#39;3 (UTR) 10. לכן, cDNAs מוכנים מ-RNA הכל על ידי שעתוק לאחור באמצעות פריימרים אקראיים במקום oligo-DT primers. זמן אמת מבחני ה-PCR עם פריימרים ספציפיים כל תת H1 (טבלה 1) מבוצעות כדי להשיג מדידה כמותית מאוד של רמות ה-mRNA של תת H1 הפרטs. הביטוי של גנים משק מנותחים כפקדי עבור נורמליזציה. השפע היחסי של החלבונים של כל תת H1 ו ההיסטונים הליבה מתקבל דרך הפוכה כרומטוגרפיה ביצועים גבוהים נוזלי שלב (RP-HPLC) ניתוח של ההיסטונים הכל המופקים בתאי יונקים 11-13. שיטת HPLC ותנאי elution המתואר כאן נותנים הפרדות אופטימלי של תת H1 עכבר. על ידי לכימות פרופיל HPLC, אנו מחשבים את חלקם היחסי של תת H1 הפרט בתוך המשפחה H1, כמו גם לקבוע את H1 יחס הנוקלאוזום בתאים.

Watch this Scientific Journal Video about Expression Analysis of Mammalian Linker-histone Subtypes at JoVE.com

Expression Analysis of Mammalian Linker-histone Subtypes

אנו מתארים סדרה של מבחני לנתח את רמות הביטוי של ההיסטונים מקשר H1. ה-mRNA של גנים בודדים H1 נמדדים באופן כמותי על ידי שעתוק אקראי מבוסס הפוך פריימר ואחריו בזמן אמת PCR, ואילו חלבון כימות של ההיסטונים H1 מושגת על ידי ניתוח HPLC.

הביטוי ניתוח Linker-היסטון תת סוגים יונקים

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

expression-analysis-of-mammalian-linker-histone-subtypes

Research

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Biology

Bacterial Gene Expression Analysis Using Microarrays

Gene Expression חיידקית ניתוח שימוש microarrays

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

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

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

הכנת ראשי בתרביות תאים hematopoietic ממח העצם Murine עבור electroporation

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

הכתמה של חלבונים עם ג&#39;ל Coomassie G-250 ללא חומצה אצטית ממס אורגני

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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.

שימוש ג&#39;ין Pulser מערכת MXcell electroporation כדי Transfect תאים ראשוניים עם יעילות גבוהה

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.

שימוש מונה תא אוטומטי כדי לפשט את הביטוי מחקרים ג&#39;ין: siRNA מציאה הביטוי IL 4-Gene תלויים תאים 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 ...

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.

ניתוח תיקון DNA פעמיים גדיל Break (DSB) בתאי יונקים

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.

Mutagenesis וניתוח פונקציונלי של תעלות יונים הביעו Heterologously בתאי יונקים

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 ...

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.

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.

אנליזה של קינטיקה mRNA גרעיני ייצוא בתאי יונקים ידי Microinjection

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

In vitro Reconstitution of the Active T. castaneum Telomerase

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

In vitro Reconstitution of the Active T. castaneum Telomerase

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

ב הכינון במבחנה של ט פעילים castaneum שהטלומרז

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

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.

ניתוח של עמדת מחזור התא בתאי יונקים

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