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

Este trabalho é apoiado pelo NIH concessão GM085261 e um Cancer Coalition Geórgia Prêmio Distinguished Scholar (a FA).

1. Preparação de amostras e de extração de RNA <ol><li> Antes da extracção de RNA, todas as superfícies de trabalho e pipetas deve ser limpo com etanol a 70% e tratado com RNase solução de descontaminação, tais como a RNase Zap. Esta prática reduz as possibilidades de RNase contaminação e degradação do ARN. Usar luvas para todos os procedimentos. </li><li> Para extrair o RNA a partir de tecido do rato, dissecar o órgão de interesse a partir do rato sacrificados, e lavar o tecido em fosfato gelada sal...

<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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linker+histone+H1+regulates+specific+gene+expression+but+not+global+transcription+in+vivo.">Linker histone H1 regulates specific gene expression but not global transcription in vivo.</a> Cell. 86, 475-483 (1996).</li><li>Alami, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+linker-histone+subtypes+differentially+affect+gene+expression+in+vivo.">Mammalian linker-histone subtypes differentially affect gene expression in vivo.</a> Proc. Natl. Acad. Sci. 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>

O conjunto de ensaios aqui apresentados permitir uma análise abrangente dos níveis de expressão de mamífero vinculador subtipos histonas. Adequadamente projetados qRT-PCR ensaios fornecem medições altamente sensível e precisa de mensagens de RNA a partir de quaisquer genes de mamíferos histona H1. A parte crítica de qRT-PCR ensaios para linker genes subtipo histona é a preparação de ADNc utilizando o iniciador aleatório baseado transcrição reversa. mRNA de genes mais histona, incluindo genes mais H1, não...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> RNase Zap </td><td> Applied Biosystems </td><td> AM9780 </td></tr><tr><td> Reagente Trizol </td><td> Invitrogen </td><td> 15596-018 </td></tr><tr><td> SuperScriptIII </td><td> Invitrogen </td><td> 18080-051 </td></tr><tr><td> Etanol absoluto </td><td> Fisher Scientific </td><td> BP2818-4 </td></tr><tr><td> QI SYBR Green </td><td> Bio-Rad </td><td> 170-8880 </td></tr><tr><td> Kit Mini RNeasy </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> Desoxirribonuclease I </td><td> Sigma </td><td> AMP-D1 </td></tr><tr><td> Microseal placa de 96 poços de PCR </td><td> Bio-Rad </td><td> MSP-9605 </td></tr><tr><td> Auto-colantes &#39;B&#39; Microseal </td><td> Bio-Rad </td><td> MSB-1001 </td&gt; </tr><tr><td> Sacarose </td><td> Organics Agros </td><td> AC40594 </td></tr><tr><td> Fosfato de sódio dibásico hepta-hidratado (Na 2 HPO 4 · 7H 2 O) </td><td> Fisher Scientific </td><td> BP332 </td></tr><tr><td> Cloreto de sódio (NaCl) </td><td> Americana Bioanalytical </td><td> AB01915 </td></tr><tr><td> Hepta-di-hidrogenofosfato de sódio (NaH 2 PO 4 · 7H2O) </td><td> Fisher Scientific </td><td> BP-330 </td></tr><tr><td> HEPES </td><td> Fisher Scientific </td><td> BP310 </td></tr><tr><td> Completa Mini proteinase comprimido coquetel inibidor </td><td> Roche Applied Science </td><td> 11836153001 </td></tr><tr><td> EDTA </td><td> Sigma </td><td> E-5134 </td></tr><tr><td> Fluoreto de Phenylmethanesulfonyl (PMSF) </td><td> Americana Bioanalytical </td><td> AB01620 </td></tr><tr><td> Nonidet-40 (NP-40) </td&gt; <td> Americana Bioanalytical </td><td> AB01425 </td></tr><tr><td> Cloreto de potássio (KCl) </td><td> Fisher Scientific </td><td> BP366 </td></tr><tr><td> Tris [hidroximetil aminometano] </td><td> Americana Bioanalytical </td><td> AB02000 </td></tr><tr><td> Cloreto de magnésio (MgCl2) </td><td> Fisher Scientific </td><td> BP214 </td></tr><tr><td> Ácido sulfúrico (H2SO4) </td><td> VWR </td><td> VW3648-3 </td></tr><tr><td> Hidróxido de amónio (NH4OH) </td><td> Organics Agros </td><td> AC42330 </td></tr><tr><td> Bradford Protein Assay </td><td> Bio-Rad </td><td> 500-0001 </td></tr><tr><td> Acetonitrila </td><td> EMD </td><td> AX0145-1 </td></tr><tr><td> Ácido trifluoroacético (TFA) </td><td> JTBaker </td><td> 9470-01 </td></tr></tbody></table>

expression analysis of mammalian linker-histone subtypes

Histona H1 Linker liga-se à partícula núcleo nucleossomo e DNA linker, facilitando o dobramento da cromatina em estrutura de ordem superior. H1 é essencial para o desenvolvimento de mamíferos 1 e regula a expressão do gene específica in vivo 2-4. Entre as proteínas altamente conservadas histona, a família de histonas linker H1 é o grupo mais heterogénea. Existem 11 subtipos H1 em mamíferos que são diferencialmente regulados durante o desenvolvimento e em diferentes tipos celulares. Estes subtipos H1 incluem 5 somática H1s (H1a-e), o H1 substituição 0, 4 subtipos de células germinativas H1 específicos, e H1x 5. A presença de subtipos H1 múltiplas que diferem na afinidade de ligação ao ADN ea capacidade de compactação cromatina 6-9 fornece um nível adicional de modulação da função da cromatina. Assim, a análise quantitativa da expressão subtipos H1 individuais, tanto de ARNm e as proteínas, é necessário para melhor compreensão da regulação de maiorestrutura de ordem cromatina e função. Descrevemos aqui um conjunto de ensaios concebidos para analisar os níveis de expressão dos subtipos H1 individuais (Figura 1). a expressão do mRNA de genes de variantes diferentes H1 é medido por um conjunto de altamente sensíveis e quantitativa transcrição reversa PCR (qRT-PCR), que são mais rápida, mais precisa e requerem amostras muito menos em comparação com a abordagem alternativa de análise de Northern blot. Ao contrário da maioria outras mensagens de ARNm celulares, mRNAs para os genes mais histona, incluindo a maioria dos genes H1, ​​carecem de uma cauda poliA de comprimento, mas contêm uma estrutura de haste loop-na extremidade 3 &#39;região não traduzida (UTR) 10. Portanto, cDNAs são preparados a partir de RNA total por transcrição inversa utilizando iniciadores aleatórios, em vez de oligo-dT iniciadores. Os ensaios em tempo real de PCR com primers específicos para cada subtipos H1 (Tabela 1) são realizados para obter medições altamente quantitativa dos níveis de mRNA de subtipo H1 indivíduos. A expressão de genes de limpeza são analisados ​​como controlos para a normalização. A abundância relativa de proteínas de cada subtipo H1 e histonas núcleo é obtido através de cromatografia líquida de fase reversa de alto desempenho (RP-HPLC) análise de histonas total extraído de células de mamíferos 11-13. O método de HPLC e as condições de eluição descrito aqui dar separações óptimas de subtipos H1 do rato. Pela quantificação do perfil de HPLC, calcula-se a proporção relativa de subtipos H1 individuais dentro da família H1, bem como determinar a H1 a razão de nucleossomas nas células.

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

Expression Analysis of Mammalian Linker-histone Subtypes

Nós descrevemos um conjunto de ensaios para analisar os níveis de expressão de histonas linker H1. mRNA de genes H1 individuais são quantitativamente medido por acaso a transcrição reversa com base iniciador seguido por PCR em tempo real, enquanto que a quantificação de proteína de histonas H1 é conseguido através da análise por HPLC.

Análise de Expressão de mamíferos Linker-histona Subtipos

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

JoVE Journal

Biology

Bacterial Gene Expression Analysis Using Microarrays

Análise de Expressão Gênica Utilizando bacteriana Microarrays

Biologia

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.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

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.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

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.

Usando o Sistema de Eletroporação Gene Pulser MXcell para transfectar pilhas com Alta Eficiência

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.

Usando um contador de células automatizadas para simplificar Estudos da Expressão Gênica: Knockdown siRNA de Expressão IL-4 Gene Dependente em células 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.

Análise de DNA dupla fita-Break Repair (DSB) em células de mamíferos

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.

Mutagênese e Análise Funcional de canais iônicos Heterologously Expresso em células de mamíferos

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.

Análise de Cinética mRNA Nuclear Exportação em células de mamíferos por microinjeção

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.

Na reconstituição in vitro da atividade T. Telomerase castaneum

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.

Análise da posição do ciclo celular em células de mamíferos

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