Sandler Asma base Research Center (SABRE) Sandler Foundation

Parte 1: Transfection di HEK 293 cellule di produrre lentivirus * 24 ore prima della trasfezione, piatto 2,5 X 10 6 celle in un piatto di 10 centimetri per una confluenza del 50-70% il giorno successivo. <ol><li> Avviare questo protocollo preparando il DNA con la quale si verrà poi trasfezione proprio le cellule per fare virus. In primo luogo, diluire FuGENE 6 Transfection Reagent Roche, un reagente lipidi che compongono le cellule prendono il DNA. In un tubo da 1,5 ml, pipetta 30μl FuGENE 6 in 600ul mezzo privo di siero (SFDMEM). Fare attenzione a non lasciare FuGENE toccare il lato del tubo in condizioni di fornitura nel mezzo. Lasciate che questo FuGENE e mescolare SFDMEM e incubare a temperatura ambiente per 5 minuti. </li><li> Nel tappo del tubo, mescolare 4 microgrammi lentivirale plasmide con 4 mg ogni 3 ° generazione di vettori di imballaggio virale (pMDL, pRSV e pVSV-G). Il plasmide lentivirale contiene il DNA del virus si inserisce nel genoma di ogni cellula che infetta, mentre i vettori di imballaggio contengono geni per tutte le altre proteine ​​necessarie per fare un lentivirus. </li><li> Chiudere il coperchio e mescolare invertendo il tubo di un paio di volte. </li><li> Incubare per 15-20 minuti a temperatura ambiente. </li><li> Pipettare intero contenuto del tubo in 293 piastre. Mescolare delicatamente piastra inclinazione avanti e indietro. </li><li> Ritorno alle cellule di dell&#39;incubatore e consentire la produzione virale di proseguire per 48-96 ore prima del raccolto. </li><li> Durante l&#39;incubazione, le 293 cellule trascrivere il DNA di interesse da parte del plasmide lentivirale, creando una copia di RNA del costrutto lentivirali. Hanno anche trascrivere e tradurre i geni nei vettori imballaggio in proteine ​​virali. Queste proteine ​​procedere a fare virus contenente la versione di RNA del costruire lentivirali, che il virus si riverseranno trascrivere e inserire nel genoma di tutte le cellule che infetta. </li></ol> Parte 2: Harvest lentivirali <ol><li> Dopo il periodo di incubazione, le cellule prendono fuori dell&#39;incubatore e utilizzare un usa e getta, siringa da 10 ml per rimuovere il supernatante, che ora contiene il virus. Ci saranno circa 10 ml di sopranatante. </li><li> Collegare un filtro 0.45μM siringa per la punta e filtrare il virus in un tubo Ultracentrifuga Beckman. Il filtro consente solo i virus, e non le cellule di passare. </li><li> Prima di gettare, incubare tutte le cellule che producono virus, piastre di coltura, siringhe e filtri nel 10% di candeggina per 45 minuti. </li></ol> Parte 3: Concentrazione lentivirali tramite ultracentrifugazione <ol><li> Carico provette in SW-41Ti o SW-28 secchi rotore. </li><li> Usa DMEM senza siero per bilanciare tutti i virus contenenti tubi a 0,2 g all&#39;interno. </li><li> Tenuta chiusa secchi rotore e agganciarli sul rotore prima di mettere dentro il rotore ultracentrifuga </li><li> Spin il virus per 90 minuti in un&#39;ultracentrifuga a 4 ° C a 25.000 giri al minuto in un SW-41Ti rotore o 24.000 rpm in un rotore SW-28. </li><li> In una cappa colture di tessuti, invertire i tubi a testa in giù a versare il surnatante in un contenitore contenente il 10% di candeggina. </li><li> Utilizzare un Kimwipe per assorbire il liquido in eccesso in tutto il pellet e invertire il tubo in un rack conveniente per non più di cinque minuti. </li><li> Per risospendere il pellet virale, utilizzare una punta filtro per aggiungere 100 ul di PBS sterile per il tubo, poi pipetta su e giù per circa 20 volte. </li><li> Lasciare il virus a 4 ° C durante la notte per completare la risospensione. Si può memorizzare prepara virale a 4 gradi per circa 1 settimana a -80 gradi per un massimo di un anno. Ricordate di trattare tutti i tubi vuoti con il 10% di candeggina prima di smaltimento. </li><li> Se si intende utilizzare la preparazione virale per più di una settimana 1, fanno aliquote minime di 5-20μl per futuri esperimenti e 1 aliquota del 3 per 5μl per la titolazione. Congelare e conservare le aliquote a meno 80. </li></ol> Parte 4: Titolazione lentivirali in citometria a flusso <ol><li> Il passo successivo consiste nel determinare il titolo virale mediante citometria di flusso. Questo metodo funziona solo nei casi in cui il plasmide lentivirale contiene un gene fluorescente reporter. L&#39;idea è che quando il virus infetta una cellula, introduce il DNA lentivirale plasmide nel genoma di quella cella. La cellula infettata poi trascrivere e tradurre i geni che hai incluso nel plasmide lentivirale, e se hai incluso un gene fluorescente giornalista, le cellule infette si fluorescenza. Il livello di fluorescenza rappresenta la potenza, o titolo, del virus. </li><li> Per ogni titolo virale di essere titolati, diluire 3 ml di virus concentrati in 1,5 ml di mezzi completi (DMEM, pen-streptococco, glutammina, FBS). </li><li> In pozzi 1-6 in una singola riga di una piastra 96 ​​pozzetti, eseguire le seguenti diluizioni seriali: a tutti i 6 pozzi aggiungere 100 ml di DMEM completo dei media. Per il primo e aggiungere 100μl di media con nessun virus (questo è il vostro controllo senza macchia), al secondo e aggiungere 100 ml di virus diluito (che è equivalente a 1 ml di virus diluito), al terzo e aggiungere 100 uldel mix dal secondo pozzo, al quarto e 100 ml dal 3 così, al quinto e aggiungere 100μl dal pozzo 4, e al sesto e aggiungere 100 microlitri dal 5 bene. Finalmente prendere 100ul fuori dal pozzo 6 e scartare in candeggina. Una diluizione seriale è stata effettuata in cui ogni bene è la metà del volume del virus rispetto a quella precedente. </li><li> Portare il volume totale in tutti i pozzetti di 200μl. Notare che questi sono diluizioni solo suggerito, che dovrebbe funzionare bene per la titolazione dei virus più concentrata. Se il virus è bassa del titolo, o non concentrata che si possano desiderare per regolare il diluizioni. </li><li> Trattare qualsiasi virus inutilizzato diluito con 10% di candeggina per 45 minuti prima di scartare in un contenitore per rifiuti a rischio biologico. </li><li> Aggiungi ~ 15.000 sano, attivamente dividendo 293 cellule per ciascuno dei 6 pozzi. Un piatto unico a 96 e può essere usato per titolo 16 preparazioni virali. </li><li> Ritorno alle cellule di grado incubatore 37 con 5% di CO 2 e permettere l&#39;infezione di procedere per almeno 48 ore. </li><li> Dopo l&#39;incubazione, le cellule vengono analizzate per l&#39;espressione giornalista (nel nostro caso fluorescenza) con un citometro a flusso. Particelle virali sono quantificato mediante la percentuale di cellule fluorescenti per bene. </li></ol> Calcolare il numero di unità infettive per ml con la seguente formula: <div style="text-align:center;"> (Numero di cellule per bene il giorno di infezione delle cellule giornalista X positivo) X 1000 ______________________________________________________ ml di vettore aggiunto al bene </div> Parte 5: Risultati Rappresentante: Dopo una di 48 ore dopo la trasfezione periodo di incubazione, la piastra di 293 cellule dovrebbe essere di circa il 90% fluorescenti. Un&#39;alta percentuale di cellule fluorescenti è indice di una elevata percentuale di cellule che producono virus. Dopo la centrifugazione, il pellet virale è appena visibile quando risospendere. Questo è normale come il pellet è chiara e molto piccolo. Nell&#39;interpretazione dei risultati citofluorimetro, si noti che si può raggiungere solo titolo calcoli precisi quando infetta (fluorescenti), le cellule hanno ricevuto più di una integrazione virale per cellula. Al fine di garantire che questo è il caso, utilizzare le cellule con il tasso di infezione &lt;15% per i calcoli. Cellule con tassi di infezione più elevati hanno probabilmente ricevuto più integrants virale per cellula. Le diluizioni suggerito al punto 2 dovrebbe produrre diversi campioni all&#39;interno di questa gamma di infezione. Idealmente, usare diversi campioni di generare una trama di titolazione (linea retta) da cui si può calcolare il titolo finale, ma si può calcolare titolo utilizzando un unico campione, se necessario. Ci si può aspettare titolo di 1,0 x 10 7 TU / ml per un-concentrato virus e 1,0 x 10 8 TU / ml per virus concentrati.

<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>

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<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>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

lentivirus production

Interferenza dell&#39;RNA (RNAi) è un sistema di silenziamento genico nelle cellule viventi. In RNAi, i geni omologhi in sequenza a breve RNA interferenti (siRNA) sono ridotti al silenzio a livello post-trascrizionale di stato. RNA tornante breve, precursori di siRNA, può essere espressa tramite lentivirus, consentendo RNAi in una varietà di tipi di cellule. Lentivirus, come il virus di immunodeficienza umana, sono in grado di infettare sia che divide e non divisione delle cellule. Ci descrivono una procedura che a lentivirus pacchetto. Imballaggio si riferisce alla preparazione di virus competente da vettori del DNA. Lentivirali sistemi di produzione vettoriali sono basati su un sistema &#39;split&#39;, in cui il genoma virale naturale è stata suddivisa in costrutti individuali plasmide helper. Questa divisione dei diversi elementi virale in quattro vettori separati diminuisce il rischio di creare una replica-virus in grado di ricombinazione casuale del genoma lentivirali. Qui, un vettore contenente i shRNA di interesse e di tre vettori imballaggio (p-VSVG, pRSV, pMDL) sono transitoriamente trasfettate in 293 cellule umane. Dopo almeno un periodo di 48 ore di incubazione, il surnatante contenente virus viene raccolto e concentrato. Infine, titolo del virus è determinata dalla giornalista (fluorescenti), espressione con un citometro a flusso.

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

Lentivirus Production

Per rendere lentivirus, vettori di DNA sono transfettate in 293 cellule umane. Dopo la raccolta e concentrando il surnatante, titolo del virus è determinata dalla espressione fluorescenza con un citometro a flusso.

Lentivirus Produzione

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 Views.  University of California, San Francisco - UCSF. Per rendere lentivirus, vettori di DNA sono transfettate in 293 cellule umane. Dopo la raccolta e concentrando il surnatante, titolo del virus è determinata dalla espressione fluorescenza con un citometro a flusso.

Video: 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.

Dimostrazione tecnica di ibridazione Whole Genome Array Comparative Genomic

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 ...

Ricerca

Biologia

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.

Inserire grande Ambientale Genomic Produzione Biblioteca

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.

Generazione di cellule staminali pluripotenti indotte da fibroblasti umani Riprogrammare con il Stemgent Lentivirus umani TF 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.

Un protocollo per la produzione di KLRG1 tetramero

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.

Estrazione DNA da materiale in paraffina per le analisi genetiche ed epigenetiche

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.

Trasduzione di cellule umane con Polymer-complessato Lentivirus Ecotropic per Enhanced biosicurezza

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.

Il MultiBac Protein Complex Piattaforma di produzione al 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.

Produzione di Haploid Zebrafish embrioni da<em&gt; In Vitro</em&gt; Concimazione

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-base Espressione Proiezione di proteine ​​di membrana 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.

La produzione di DNA a doppia elica 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 ...