Name: transient expression of proteins by hydrodynamic gene delivery in mice
Uploaded: 2014-05-05T16:30:00
Description: Watch this Scientific Journal Video about Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice at JoVE.com

Vi takker Xia Liu (Regeneron Pharmaceuticals) for å begynne å lære oss den HGD teknikk og Dr. Russell Thomson (Albert Einstein College of Medicine) for sin vedvarende veiledning i ulike aspekter av teknologien. Dette arbeidet ble finansiert av Hunter College, CUNY starte opp midler og NSF Brød award IOS-1249166. Vi takker NYULMC Histopathology Kjerne NYUCI senter Support Grant, NIH / NCI 5 P30CA16087-31 for histologi på mus lever.

Alle eksperimenter er beskrevet her ble godkjent av Institutional Animal Care og bruk komité Hunter College, City University of New York. En. Utarbeidelse av endotoksin-fri plasmid DNA <ol><li> Plukk en enkelt koloni av bakterier inneholdende genet av interesse i en pattedyr ekspresjonsvektor fra en nylig stripete selektiv plate. </li><li> Følge anbefalingene som finnes i håndboken av et kommersielt tilgjengelig endotoxin-fritt plasmid rensing kit for dyrking og høsting bakterier. </li...

<ol>
	<li>Liu, F., Song, Y., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamics-based+transfection+in+animals+by+systemic+administration+of+plasmid+DNA.">Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA.</a> Gene Ther. 6, 1258-1266 (1999).</li><li>Zhang, G., Budker, V., Wolff, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+levels+of+foreign+gene+expression+in+hepatocytes+after+tail+vein+injections+of+naked+plasmid+DNA.">High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA.</a> Hum Gene Ther. 10, 1735-1737 (1999).</li><li>Miao, C. H., Thompson, A. 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Når utført riktig, er HGD en bemerkelsesverdig trygg og effektiv måte å transgenet levering. De kritiske trinn til vellykket HGD er: 1) å levere den riktige mengde av DNA i et stort volum av saltløsningsbærer 2) inn i halevenen på mus 3) på mindre enn 8 sekunder. Selv om prosessen med injeksjon seg unektelig krever litt fingerferdighet, suksessen av denne seks-andre prosedyre ofte ligger i grundige forberedelser av eksperimentet. Mengden av pDNA nødvendig for å oppnå maksimal gen...

Siden sin første beskrivelse av Liu et al. Og Zhang et al., Har hydrodynamisk genet levering (HGD) blitt et uvurderlig verktøy for å studere gen-funksjon hos gnagere modellsystemer 1, 2. Teknikken innebærer en rask injeksjon (5-7 sekunder) fra et stort volum (8-12% av kroppsvekten) av oppløsningen inn i halevenen til mus for å lette opptaket av plasmid-DNA av cellene i målorganene 1, 2. Disse forhold fører til robust genekspresjon i leveren og mindre genekspresjon i nyren, milt, lunge og hjerte. Injeksjon av 10 mikrogram pCMV-LacZ plasmid kan transfektere så mye som 40% av hepatocytter, gjør HGD den mest effektive, ikke-viral, in vivo genet levering metode for å date en. I motsetning til virusbærere, er enkel å tilberede pDNA, ikke framprovosere en immunrespons i gnager host tre og ikke utgjør en helserisiko ved å rekombinere med sluttenogenous virus. I tillegg, siden den DNA-molekyler som leveres av HGD ikke trenger emballasje, er denne metoden er egnet for levering av bakterielle kunstige kromosomer (BAC) så store som 150 kb 4. Andre typer av molekyler som har blitt levert av en hydrodynamisk fremgangsmåte omfatter RNA 5-10, Morpholinos 11, proteiner 12, 13 og andre små molekyler 12, 14. Fordelene og ulempene ved HGD fremfor andre leveringsmetoder har vært diskutert i gode anmeldelser i litteraturen 15-20 og en rekke forfattere har gitt en detaljert beskrivelse av prosedyren 21-23. Vi presenterer transgener i mus ved HGD er trygg og effektiv 1-3, 24 og metoden har vært brukt i rotter med sammenlign suksess 25. Med visse modifikasjoner, har proof-of-concept eksperimenter utført i kyllinger 26, Rabbits 27 og griser 28, selv om den in vivo-anvendelsen av denne teknikk i større dyr er fortsatt en utfordring. Når du bruker denne metoden, er en annen vanlig begrensning at mange av de tilgjengelige pattedyr ekspresjonsvektorene mangler komponenter for å oppnå en vedvarende, høy grad av genuttrykk. Ved hjelp av en pCMV-Luc plasmid, genuttrykk i målet organer er tydelig så tidlig som ti minutter etter HGD, derimot, faller den første, høyt uttrykk nivået kraftig i første uke etter injeksjonen en. Long-term transgen ekspresjon er mulig, avhengig av promoter-og intron anvendes i plasmid utforming 3, 24 er imidlertid opprettholdelsen av høynivå genekspresjon ofte krever gjentatte injeksjoner. Av denne grunn, kan HGD være mindre egnet til å studere kroniske sykdommer som er et resultat av langvarig eksponering for skadelige proteiner eller proteinprodukter. Med disse begrensningene, er HGD en usedvanlig kraftig verktøy for å studere den potensiale rolle av et gen og effekten av det mutanter in vivo, så vel som de terapeutiske effekter og regulering av proteiner og for å etablere dyremodeller av sykdom (for gjennomgang, se 15). For eksempel kan HGD benyttes for å knytte funksjonen til domener og aminosyrer i proteiner ved individuelt å innføre forskjellige gen-konstruksjoner i mus som har de respektive gener slått ut. Videre kan denne teknikken bli brukt i en hvilken som helst muse belastning. Denne protokollen beskriver HGD i mus med fokus på de tekniske aspektene er nødvendig for å oppnå vellykket transfeksjon: riktig nål innsetting i venen, injeksjonsvolumet og hastigheten på levering. Anvendelsen av denne fremgangsmåte er vist i en musemodell av afrikansk trypanosomiasis, en dødelig sykdom hos mennesker og husdyr 29, 30. Mens flere arter av trypanosomer forårsake sykdom hos husdyr, kan de ikke forårsaker sykdom hos mennesker på grunn av medfødte immunkomplekser i blood kalt trypanosom lytiske faktorer (TLFs) 29, 31, 32. Disse poredannende, high-density lipoproteiner (HDL) inneholder to unike, primat-spesifikke proteiner:. HPR, liganden, som letter opptaket av TLFs inn trypanosomer, og APOL-I, lytisk komponenten 31, 33-38 Trypanosoma brucei rhodesiense er i stand til å infisere mennesker på grunn av ekspresjon av et serum resistens-assosiert protein (SRA) som binder seg til og nøytraliserer human APOL-I 34, 39. Baboon TLF er ikke nøytralisert av SRA grunn av sin avvik APOL-Jeg protein 40. Som rapportert tidligere, ved hjelp av en pattedyr uttrykk vektor (pRG977), transgene uttrykk for bavian TLF komponenter i mus ensidige beskyttelse mot menneske-infeksiøs trypanosomer 40. De representative data presentert her viser hvordan hydrodynamisk genet levering kan anvendes for å studere de terapeutiske virkninger av et protein.

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			<td height="21" style="height:21px;width:216px;">Alcohol Prep Wipe</td>
			<td style="width:113px;">Webcol</td>
			<td align="right" style="width:119px;">6818</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AST kit, Amplite Colorimetric</td>
			<td style="width:113px;">AAT Bioquest</td>
			<td align="right" style="width:119px;">13801</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical Tube, 50 ml</td>
			<td style="width:113px;">BD Falcon</td>
			<td align="right" style="width:119px;">352070</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:113px;">Qiagen</td>
			<td align="right" style="width:119px;">12362</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KimWipes</td>
			<td style="width:113px;">KIMTECH</td>
			<td align="right" style="width:119px;">34120</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Mouse Tail Illuminator</td>
			<td style="width:113px;">Braintree Scientific</td>
			<td style="width:119px;">MTI RST</td>
			<td style="width:167px;">Or equivalent</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Needle, 20 Gx 1 (0.9mmx25mm)</td>
			<td style="width:113px;">BD</td>
			<td align="right" style="width:119px;">305175</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Needle, 27 Gx 1/2 (0.9mmx25mm)</td>
			<td style="width:113px;">BD</td>
			<td align="right" style="width:119px;">305109</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">pRG977 (mammalian expression vector)</td>
			<td style="width:113px;">Regeneron Pharmaceuticals</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Material Transfer Agreement required</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sodium Chloride, 0.9% INJ USP 10 ml, Hospira&#160;</td>
			<td style="width:113px;">Fisher</td>
			<td style="width:119px;">NC9054335</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Syringe, 3ml, Luer-Lok Tip</td>
			<td style="width:113px;">BD</td>
			<td align="right" style="width:119px;">309657</td>
			<td style="width:167px;">Luer Lock necessary to withstand pressure from HGD</td>
		</tr>
	</tbody>
</table>

transient expression of proteins by hydrodynamic gene delivery in mice

Effektiv uttrykk av transgener i vivo er av avgjørende betydning i å studere gen-funksjon og utvikle behandlingsmetoder for sykdommer. I løpet av de siste årene, har hydrodynamisk genet levering (HGD) dukket opp som en enkel, rask, trygg og effektiv metode for å levere transgener til gnagere. Denne teknikk er avhengig av den kraft som genereres av den raske injeksjon av et stort volum av fysiologisk løsning for å øke permeabiliteten av cellemembraner av perfusert organer og derved levere DNA inn i celler. En av de viktigste fordelene med HGD er muligheten for å innføre transgener inn i pattedyrceller ved bruk av nakent plasmid DNA (pDNA). Vi presenterer en eksogen genet ved hjelp av et plasmid er minimal arbeidskrevende, svært effektiv og, i motsetning til virus-bærere, bemerkelsesverdig trygg. HGD ble opprinnelig brukt for å levere gener i mus, er det nå brukes til å levere et bredt spekter av substanser, inklusive oligonukleotider, kunstige kromosomer, RNA, proteiner og små molekyler i mus, rotterog, i begrenset grad, andre dyr. Denne protokollen beskriver HGD i mus og fokuserer på tre viktige aspekter ved den metoden som er kritiske til å utføre prosedyren vellykket: korrekt innsetting av nålen inn i venen, volumet av injeksjon og hastigheten på levering. Eksempler er gitt for å vise anvendelsen av denne fremgangsmåte for transient ekspresjon av to gener som koder for utskilte, primat-spesifikke proteiner, apolipoprotein LI (APOL-I) og haptoglobin-relatert protein (HPR).

Riktig plassering av nålen inn i halevenen på mus (figur 1) er en forutsetning for en vellykket levering av et transgen av hydrodynamikk-basert transfeksjon. Ofte er den mest krevende delen av teknikken, er imidlertid at bibehold av nålen inne i halevenen uten bevegelse, slik at hele volumet av injeksjon kan leveres i løpet av en 6-8 s periode. Mindre feil under injeksjonsprosessen kan føre til vesentlig redusert transfeksjon effektivitet og proteinekspresjon. Derfor er det viktig å overvåke suks...

Watch this Scientific Journal Video about Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice at JoVE.com

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

In vivo transfeksjon av naken DNA ved hydrodynamisk genet levering introduserer gener inn i vevet til et dyr med minimal inflammatorisk respons. Tilstrekkelige mengder av gen-produktet er generert slik at gen-funksjon og regulering, så vel som proteinstruktur og funksjon kan analyseres.

Gående uttrykk av proteiner ved Hydrodynamisk Gene Levering i Mus

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

transient-expression-of-proteins-by-hydrodynamic-gene-delivery-in-mice

Research

JoVE Journal

Biology

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.

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.

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.

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.

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 ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

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.

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

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Effektiv Agroinfiltration of Plants for High-level Forbigående Expression of rekombinante proteiner

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analyse av global RNA syntese på celle-nivå etter Hypoksi

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Måling av Metabolic Rate i<em&gt; Drosophila</em&gt; Hjelp respirometri

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.