Sandler Asthma Basic Research Center (SABRE) Sandler Foundation

Teil 1: Transfektion von HEK 293 Zellen zu Lentiviren Produce * 24 Stunden vor der Transfektion Platte 2,5 x 10 6 Zellen in eine 10 cm Schüssel für eine Konfluenz von 50-70% am nächsten Tag. <ol><li> Starten Sie dieses Protokoll durch die Vorbereitung der DNA, mit denen man später transfizieren wird man die Zellen auf Viren zu machen. Zuerst verdünnten FuGENE 6 Transfektionsreagenz Roche, ein Lipid-Reagenz, dass Zellen nehmen DNA zu machen. In einem 1,5 ml Röhrchen, Pipette 30μl FuGENE 6 in 600ul serum-freiem Medium (SFDMEM). Achten Sie darauf, nicht zulassen FuGENE Touch der Seite der Röhre, wie sie in das Medium abgegeben wird. Lassen Sie diese FuGENE und SFDMEM mischen und inkubieren bei Raumtemperatur für 5 Minuten. </li><li> In die Kappe des Röhrchens mischen 4 ug lentiviralen Plasmid mit 4 g je 3 rd Generation viraler Vektoren Verpackung (pMDL, pRSV und pVSV-G). Die lentiviralen Plasmid enthält die DNA des Virus wird in das Genom jeder Zelle infiziert es einzufügen, während die Verpackung Vektoren Gene für alle anderen Proteine ​​benötigt, um ein Lentivirus make enthalten. </li><li> Schließen Sie den Deckel und mischen durch Invertieren des Röhrchens ein paar mal. </li><li> Inkubieren 15-20 Minuten bei Raumtemperatur. </li><li> Pipette gesamte Inhalt des Röhrchens in 293 Teller. Vorsichtig mischen durch Kippen Platte hin und her. </li><li> Bringen Sie die Zellen in den Inkubator und ermöglichen virale Produktion für 48-96 Stunden vor der Ernte weiter. </li><li> Während der Inkubation transkribieren die 293-Zellen die DNA von Interesse aus der lentiviralen Plasmid, wodurch eine RNA-Kopie des lentiviralen konstruieren. Sie haben auch transkribieren und übersetzen die Gene in der Verpackung Vektoren in virale Proteine. Diese Proteine ​​gehen, um Viren, die die RNA-Version Ihres lentivirale Konstrukt, das das Virus übertragen umkehren wird und in das Genom aller Zellen, infiziert zu machen. </li></ol> Teil 2: Lentivirale Ernte <ol><li> Nach Inkubationszeit, nehmen Zellen aus dem Inkubator und mit einem Einweg-, 10ml Spritze mit dem Überstand, das nun den Virus zu entfernen. Es wird etwa 10 ml des Überstands werden. </li><li> Bringen Sie ein 0.45 Spritze Filter auf die Spitze und Filter des Virus in einer Beckman Ultrazentrifuge Röhre. Der Filter lässt nur Viren und nicht-Zellen passieren. </li><li> Vor dem Entsorgen, inkubieren alle Virus-produzierenden Zellen, Kultur-Platten, Spritzen und Filter in 10% Bleichmittel für 45 Minuten. </li></ol> Teil 3: Lentivirale Konzentration über Ultrazentrifugation <ol><li> Laden Zentrifugenröhrchen in SW-41Ti oder SW-28 Rotor Eimer. </li><li> Verwenden Sie serumfreiem DMEM für alle Virus-haltigen Gleichgewicht Rohre innerhalb 0.2g. </li><li> Seal geschlossen Laufschaufeln und Haken sie auf den Rotor, bevor Rotor im Inneren der Ultrazentrifuge </li><li> Spin des Virus für 90 Minuten in einer Ultrazentrifuge bei 4 ° C bei 25.000 rpm in einem SW-41Ti Rotor oder 24.000 rpm in einem SW-28 Rotor. </li><li> In einer Gewebekultur Kapuze, invertieren Röhrchen kopfüber an Überstand in einen Behälter mit 10% Bleichmittel gießen. </li><li> Verwenden Sie einen Kimwipe, um überschüssige Flüssigkeit rund um die Pellets zu absorbieren und das Röhrchen in eine bequeme Ablage für nicht mehr als fünf Minuten. </li><li> Zum erneuten Aussetzung der viralen Pellet, einen Filter verwenden, Spitze bis 100 ul sterilem PBS auf das Rohr hinzufügen, dann Pipette auf und ab etwa 20-mal. </li><li> Lassen Sie das Virus bei 4 ° C über Nacht Resuspension abzuschließen. Man kann virale preps bei 4 Grad lagern für ca. 1 Woche und bei -80 Grad bis zu einem Jahr. Denken Sie daran, alle leeren Tuben mit 10% Bleiche vor dem Wegwerfen zu behandeln. </li><li> Wenn man das virale prep länger als die 1 Woche nutzen will, um minimal Aliquots von 5-20 &amp;mgr; l für zukünftige Experimente und 1 Aliquot von 3 bis 5μl für die Titration. Freeze und speichern Sie die Aliquots bei minus 80. </li></ol> Teil 4: Lentivirale Titration mittels Durchflusszytometrie <ol><li> Der nächste Schritt ist, um das virale Titer mittels Durchflusszytometrie bestimmt. Diese Methode funktioniert nur in Fällen, in denen die lentiviralen Plasmid enthält einen fluoreszierenden Reporter-Gen. Die Idee ist, dass, wenn das Virus eine Zelle infiziert, es dem lentiviralen Plasmid-DNA führt in die Zelle das Genom. Die infizierte Zelle wird dann transkribieren und übersetzen die Gene die Sie in der lentiviralen Plasmid, und wenn Sie einen fluoreszierenden Reporter-Gen enthalten, infizierte Zellen fluoreszieren. Die Höhe der Fluoreszenz repräsentiert die Potenz oder Titer des Virus. </li><li> Für jeden viralen Bestand an titriert werden, verdünnte 3 ul konzentrierter Virus in 1,5 ml komplettem Medium (DMEM, Pen-Strep, Glutamin, FBS). </li><li> In Brunnen 1-6 in einer einzigen Zeile einer 96-Well-Platte, die folgenden Verdünnungsreihen: alle 6 Vertiefungen werden 100 ul von kompletten DMEM-Medien. Um die erste und fügen 100 &amp;mgr; von Medien ohne Virus (dies Ihr ungefärbten Kontrolle), die zweite und mit 100 ul der verdünnten Virus (das entspricht to1 ml unverdünnter Virus), den dritten und mit 100 ulder Mischung aus der zweiten und zur vierten gut 100 ml ab dem 3. gut, in den fünften und fügen 100 &amp;mgr; vom 4. gut, und die sechste und mit 100 ul aus dem 5. gut. Schließlich nehmen 100 ul aus dem 6. und und entsorgen in Bleichmittel. Eine Verdünnungsreihe wurde durchgeführt, wo jeder gut die Hälfte des Volumens des Virus im Vergleich zu dem davor. </li><li> Bringen Sie das Gesamtvolumen in alle Vertiefungen zu 200 ul. Beachten Sie, dass diese nur angedeutet Verdünnungen, die gut funktionieren sollte Titrierung konzentriertesten Viren sind. Wenn der Virus ist gering im Titer oder unkonzentriert, den Sie benötigen, um die Verdünnungen anzupassen. </li><li> Gönnen Sie unbenutzte verdünnten Virus mit 10% Bleichmittel für 45 Minuten vor dem Wegwerfen in eine biohazard Abfallbehälter. </li><li> Add ~ 15.000 gesunde, sich aktiv teilenden 293-Zellen zu jedem der 6 Vertiefungen. Ein einziger 96-Well-Platte kann auf 16 virale Titer preps werden. </li><li> Return-Zellen bis 37 Grad Inkubator mit 5% CO 2 und damit Infektion für mindestens 48 Stunden vor. </li><li> Nach der Inkubation werden die Zellen für Reporter Ausdruck (in unserem Fall Fluoreszenz) mit einem Durchflußzytometer analysiert. Virale Partikel werden durch die Prozent der fluoreszierenden Zellen pro Vertiefung quantifiziert. </li></ol> Berechnen Sie die Anzahl der infektiösen Einheiten pro ml nach folgender Formel: <div style="text-align:center;"> (Anzahl der Zellen pro Vertiefung am Tag der Infektion X-Reporter positive Zellen) X 1000 ______________________________________________________ ml Vektor hinzugefügt, um auch </div> Teil 5: Repräsentative Ergebnisse: Nach einer 48-stündigen Inkubationszeit nach der Transfektion sollten die Platte von 293 Zellen bei etwa 90% fluoreszierende werden. Ein hoher Prozentsatz der fluoreszierenden Zellen ist ein Hinweis auf einen hohen Anteil an Virus-produzierenden Zellen. Nach Zentrifugation wird das virale Pellet kaum sichtbar, wenn Resuspendieren. Dies ist normal, da das Pellet ist klar und sehr klein. Bei der Interpretation Durchflusszytometer Ergebnisse, beachten Sie, dass kann man nur erreichen, genaue Titer Berechnungen, wenn infizierte (Fluoreszenz)-Zellen haben nicht mehr als eine virale Integration pro Zelle aufgenommen. Um sicherzustellen, dass dies der Fall ist, verwenden Sie Zellen mit &lt;15% Infektionsrate für die Berechnungen. Zellen mit höheren Infektionsraten wahrscheinlich mehrere virale Integranten pro Zelle aufgenommen. Die Verdünnungen in Schritt 2 vorgeschlagen sollten mehrere Proben in diesem Bereich der Infektion ergeben. Idealerweise sollten mehrere Proben auf eine Titration Grundstück (linear-Linie), von dem Sie das letzte Titer berechnen erzeugen können, aber Sie können Titer berechnen mit Hilfe einer einzigen Probe, wenn nötig. Man kann erwarten, Titer von 1,0 x 10 7 TU / ml für un-konzentriert Virus und 1,0 x 10 8 TU / ml für konzentriertes Virus.

<ol>
	<li>Reiser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+concentration+of+pseudotyped+HIV-1-based+gene+transfer+vectors..">Production and concentration of pseudotyped HIV-1-based gene transfer vectors..</a> Gene Therapy. , 72-7211 (2000).</li><li>Dull, T., Zufferey, R., Kelly, M., Mandel, R. J., Nguyen, M., Trono, D., Naldini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Virol. 72 (11), 8463-8471 (1998).</li></ol>

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>DMEM HEK 293T cells Penicillin-streptomycin Glutamine Phosphate-buffered saline FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FuGENE 6 transfection reagent pSico mCherry pMDL pRSV pVSVG</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bleach</td>
<td>&nbsp;</td>
<td>Clorox</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10-cm cell culture dishes 96 well cell culture dishes </td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>353003</td>
<td>&nbsp;</td>
<tr>
<td>Multichannel pipette</td>
<td>&nbsp;</td>
<td>Rainin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Filtered pipette tips</td>
<td>&nbsp;</td>
<td>Rainin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5ml Eppendorf tubes Syringe Syinge filter (0.45-&mu;m)</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge tubes</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture incubator at 5% CO2</td>
<td>&nbsp;</td>
<td>Coulter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Beckman SW41T.I rotor</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Beckman ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope</td>
<td>&nbsp;</td>
<td>Coulter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FACS Array</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>&nbsp;</td>
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lentivirus production

RNA-Interferenz (RNAi) ist ein System von Gen-Silencing in lebenden Zellen. In RNAi, homologe Gene in der Reihenfolge auf short interfering RNAs (siRNA) sind in der post-transkriptionellen Staat zum Schweigen gebracht. Kurze Haarnadel-RNAs, Vorstufen von siRNA, kann mit Hilfe Lentivirus, so dass für RNAi in einer Vielzahl von Zelltypen werden. Lentiviren, wie das Human Immunodeficiency Virus, sind in der Lage zu infizieren sowohl teilende und nicht-teilenden Zellen. Wir beschreiben ein Verfahren, das zum Paket Lentiviren. Verpackung bezieht sich auf die Vorbereitung der zuständige Virus aus der DNA-Vektoren. Lentiviralen Vektor Produktion Systeme basieren auf einer &#39;split&#39; System, wo die natürlichen viralen Genoms in einzelne Helfer Plasmid-Konstrukte wurde spaltet. Diese Aufspaltung der verschiedenen viralen Elementen in vier getrennten Vektoren vermindert die Gefahr der Schaffung von ein Replikations-fähig Virus durch zufällige Rekombination des lentiviralen Genoms. Hier sind ein Vektor, der die shRNA von Interesse und drei Verpackung Vektoren (p-VSVG, pRSV, pMDL) transient in humane 293-Zellen transfiziert. Nach mindestens einer 48-stündigen Inkubationszeit wird das Virus enthaltende Überstand geerntet und aufkonzentriert. Schließlich ist Virustiter von Reporter (fluoreszierend) Ausdruck mit einem Durchflußzytometer bestimmt.

Watch this Scientific Journal Video about Lentivirus Production at JoVE.com

Lentivirus Production

Um Lentiviren, sind DNA-Vektoren in humanen 293-Zellen transfiziert. Nach der Ernte und die Konzentration der Überstand wird Virustiter mittels Fluoreszenz-Ausdruck mit einem Durchflußzytometer bestimmt.

Lentivirus Produktion

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

Research

JoVE Journal

Biology

65.6K Aufrufe.  University of California, San Francisco - UCSF. Um Lentiviren, sind DNA-Vektoren in humanen 293-Zellen transfiziert. Nach der Ernte und die Konzentration der Überstand wird Virustiter mittels Fluoreszenz-Ausdruck mit einem Durchflußzytometer bestimmt.

Serie: Lentivirus Production

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

Technische Demonstration von Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

Forschung

Biologie

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

Large Insert Environmental Genomic Bibliothek Produktion

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

Erzeugung von induzierten pluripotenten Stammzellen durch Reprogrammierung menschlichen Fibroblasten mit dem Stemgent Menschliche TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

Ein Protokoll für die Produktion von KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

DNA-Extraktion aus in Paraffin eingebettete Material für genetische und epigenetische Analysen

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

Transduktion humaner Zellen mit Polymer-Komplex ecotropen Lentivirus für Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Die MultiBac Protein Complex Produktion Platform am EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

Die Produktion von Haploid Zebrafischembryonen durch<em&gt; In-vitro-</em&gt; Düngung

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-basierte Screening Expression von Membranproteinen in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Production of Double-stranded DNA Ministrings

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ministrings possess a heightened safety profile and also efficiently deliver DNA cargo to targeted cells. Conventional DNA vectors carry undesirable prokaryotic sequences, including antibiotic resistance genes, CpG motifs, and bacterial origins of replication, which may lead to the stimulation of host immunological responses. The bioavailability of conventional DNA vectors is also compromised due to their larger molecular size. Their circular nature may also impart chromosomal integration, leading to insertional mutagenesis.
Bacterial sequences are excised from DNA minivectors, leaving only the gene of interest (GOI) and necessary eukaryotic expression elements. Our LCC DNA minivectors, or DNA ministrings, are devoid of immunogenic bacterial sequences; therefore improving their bioavailability and GOI expression. In the event of vector integration into the chromosome, the LCC DNA ministring will lethally disrupt the host chromosome, thereby removing the potentially dangerous mutant from the proliferating cell population. Consequently, DNA ministrings offer the benefits of &#39;minicircle&#39; DNA while eliminating the potential for undesirable vector integration events. In comparison to conventional plasmids and their isogenic circular covalently closed (CCC) counterparts, DNA ministrings demonstrate superior bioavailability, transfection efficiency, and cytoplasmic kinetics - they thus require lower amounts of cationic surfactants for effective transfection of target cells.
We have constructed a one-step inducible in vivo system for the production of DNA ministrings in Escherichia coli that is simple to use, rapid, and scalable.

The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings.

Die Produktion von Doppelstrang-DNA-Ministrings

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ...