Sandler Asma Básico Research Center (SABRE) Sandler Foundation

Parte 1: Transfecção de HEK 293 células para produzir Lentivirus * 24 horas antes de transfecção, placa de 2,5 X 10 6 células em um prato de 10 centímetros de uma confluência de 50-70% no dia seguinte. <ol><li> Iniciar este protocolo, preparando o DNA com o qual se vai depois de uma transfecção as células para fazer vírus. Primeiro, diluir FuGENE 6 Transfection reagente Roche, um reagente de lipídios que fazem as células ocupam DNA. Em um tubo de 1,5 ml, pipetar 30μl FuGENE 6 em meio isento de soro 600ul (SFDMEM). Tenha cuidado para não deixar FuGENE toque no lado do tubo quando é entregue ao meio. Deixe esta FuGENE e SFDMEM misturar e incubar à temperatura ambiente por 5 minutos. </li><li> Na tampa do tubo, misturar 4 mg lentiviral plasmídeo com 4 mg a cada 3 ª geração de vetores virais embalagem (pMDL, PRSV e pVSV-G). O plasmídeo lentiviral contém o DNA do vírus irá inserir no genoma de cada célula que infecta, enquanto os vetores de embalagens contêm genes para todas as outras proteínas necessárias para fazer um lentivírus. </li><li> Feche a tampa e misturar invertendo o tubo várias vezes. </li><li> Incubar 15-20 minutos em temperatura ambiente. </li><li> Pipeta conteúdo inteiro do tubo em 293 placa. Misture delicadamente a placa de inclinação para trás e para frente. </li><li> As células voltem para a incubadora e permitir que a produção viral continuar por 48-96 horas antes da colheita. </li><li> Durante a incubação, a 293 células transcrever o DNA de interesse do plasmídeo lentiviral, criando uma cópia de RNA da construção lentiviral. Eles também transcrever e traduzir os genes nos vetores de embalagens em proteínas virais. Estas proteínas continuar a fazer vírus contendo a versão do seu RNA construir lentiviral, que o vírus irá reverter transcrever e inserir no genoma de todas as células que infecta. </li></ol> Parte 2: Harvest Lentivirus <ol><li> Após período de incubação, retirar células fora da incubadora e use uma seringa descartável de 10ml, para remover o sobrenadante, que agora contém o vírus. Haverá cerca de 10ml do sobrenadante. </li><li> Coloque um filtro de seringa de 0,45 para a ponta e filtrar o vírus em um tubo de ultracentrífuga Beckman. O filtro permite que apenas os vírus, e não as células a passar. </li><li> Antes de deitar fora, incubar todas as células produtoras de vírus, placas de cultura, seringas e filtros em água sanitária 10% por 45 minutos. </li></ol> Parte 3: Concentração Lentivirus via Ultracentrifugação <ol><li> De carga em tubos de centrífuga SW-41Ti ou SW-28 baldes de rotor. </li><li> Use soro livre de DMEM para equilibrar todos os vírus contendo tubos para dentro de 0.2g. </li><li> Seal fecha baldes rotor e conectá-los em rotor antes de colocar dentro do rotor ultracentrífuga </li><li> Girar o vírus por 90 minutos em uma ultracentrífuga a 4 ° C a 25.000 rpm em um rotor SW-41Ti ou 24.000 rpm em um rotor SW-28. </li><li> Em uma capa de cultura de tecidos, inverter os tubos de cabeça para baixo para derramar sobrenadante em um recipiente contendo água sanitária a 10%. </li><li> Use uma Kimwipe para absorver o excesso de líquido ao redor do pellet e inverter o tubo em um rack conveniente para não mais que cinco minutos. </li><li> Para voltar a suspender o pellet viral, use uma ponta de filtro para adicionar 100 mL de PBS estéril ao tubo, então pipeta cima e para baixo cerca de 20 vezes. </li><li> Deixe o vírus a 4 ° C durante a noite para completar re-suspensão. Pode-se armazenar preps viral a 4 graus por cerca de uma semana e à temperatura de -80 graus por até um ano. Lembre-se de tratar todos os tubos vazios com água sanitária a 10% antes de descartar. </li><li> Se alguém pretende usar a preparação viral por mais tempo do que a semana 1, fazer alíquotas mínima de 5-20μl para futuros experimentos e uma alíquota de 3 a 5μl para a titulação. Congelar e armazenar as alíquotas de 80 de menos. </li></ol> Parte 4: Titulação Lentivirus por citometria de fluxo <ol><li> O próximo passo é determinar o título viral por citometria de fluxo. Este método só funciona nos casos em que o plasmídeo lentiviral contém um gene repórter fluorescente. A idéia é que quando o vírus infecta uma célula, ele introduz o DNA lentiviral plasmídeo no genoma dessa célula. Da célula infectada, então, transcrever e traduzir os genes que você incluiu no plasmídeo lentiviral, e se você incluiu um gene repórter fluorescente, as células infectadas ficam fluorescentes. O nível de fluorescência representa a potência, ou título, do vírus. </li><li> Para cada ação viral para ser tituladas, diluir 3 mL do vírus concentrado em 1,5 ml de meio completo (DMEM, pen-strep, glutamina, FBS). </li><li> Em poços de 1-6 em uma única linha de uma placa de 96 poços, execute as seguintes diluições em série: a todos os seis poços adicionar 100 ml de DMEM completo de mídia. Para o primeiro poço adicionar 100μl dos meios de comunicação, sem vírus (este é o seu controle sem mácula), para o segundo poço adicionar 100 ml do vírus diluído (que é para 1 ml equivalente do vírus diluído), para o terceiro também adicionar 100 ulda mistura a partir do segundo poço, para o quarto poço adicionar 100 ml do terceiro poço, para o quinto também adicionar 100μl do poço 4, e para o sexto bem adicionar 100 ul da quinta também. Finalmente assumir 100ul do poço 6 e descartar em lixívia. A diluição em série foi realizada em cada poço tem metade do volume de vírus em comparação com o anterior. </li><li> Trazer o volume total em todos os poços de 200μl. Note que estes são diluições apenas sugerido, que deve funcionar bem para titulação vírus mais concentrado. Se o vírus é baixa no título, ou não concentrada pode-se necessário ajustar as diluições. </li><li> Tratar qualquer vírus não utilizado diluído em água sanitária 10% por 45 minutos antes de descartar em um recipiente de resíduos de risco biológico. </li><li> Adicionar ~ 15000 saudável, dividindo ativamente 293 células para cada um dos 6 poços. A única placa de 96 poços pode ser usado para título 16 preps viral. </li><li> Retorno células a 37 graus incubadora com 5% de CO 2 e permitir a infecção para prosseguir durante pelo menos 48 horas. </li><li> Após a incubação, as células são analisadas para expressão repórter (no nosso caso de fluorescência), com um citômetro de fluxo. Partículas virais são quantificada pela porcentagem de células fluorescentes por poço. </li></ol> Calcule o número de unidades infecciosas por ml com a seguinte fórmula: <div style="text-align:center;"> (# De células por poço no dia de células infecção repórter X positivo) X 1000 ______________________________________________________ ml do vetor adicionado ao bem </div> Parte 5: Resultados Representante: Depois de 48 horas de transfecção pós período de incubação, a placa de 293 células deve ser em torno de 90% fluorescentes. A elevada percentagem de células fluorescentes é uma indicação de uma alta porcentagem de células produtoras de vírus. Após a centrifugação, o pellet viral é pouco visível quando ressuspensão. Isso é normal como o pellet é clara e muito pequenas. Ao interpretar os resultados citômetro de fluxo nota, que só se pode alcançar cálculos título precisos quando infectados (fluorescente), as células não receberam mais do que uma integração viral por célula. , A fim de garantir que este for o caso, use com células &lt;taxa de infecção de 15% para os cálculos. Células com maiores taxas de infecção provável recebeu vários integrantes viral por célula. As diluições sugerido no passo 2 deve render várias amostras dentro desta faixa de infecção. Idealmente, use várias amostras para gerar um gráfico de titulação (linha linear) a partir do qual você pode calcular o título final, mas você pode calcular título usando uma única amostra, se necessário. Pode-se esperar título de 1,0 x 10 7 TU / ml para un concentrado de vírus e 1,0 x 10 8 TU ml / para o vírus concentrado.

<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>
		</tr>
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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

Interferência de RNA (RNAi) é um sistema de silenciamento de genes em células vivas. Em RNAi, genes homólogos em seqüência de curto RNAs de interferência (siRNA) são silenciadas no estado pós-transcricional. RNAs hairpin curto, precursores de siRNA, podem ser expressas utilizando lentivírus, permitindo a RNAi em uma variedade de tipos de células. Lentivírus, como o Vírus da Imunodeficiência Humana, são capazes de infectar tanto divisão e não divisão das células. Vamos descrever um procedimento que a lentivírus pacote. Embalagem refere-se à preparação de vírus competentes de vetores de DNA. Sistemas de produção Lentivirus vector são baseados em um &#39;split&#39; do sistema, onde o genoma viral naturais foi dividida em construções auxiliares individuais plasmídeo. Essa divisão dos diferentes elementos viral em quatro vetores separados diminui o risco de criar um vírus de replicação com capacidade de recombinação do genoma acidental lentiviral. Aqui, um vetor contendo os shRNA de interesse e de três vetores de embalagem (p-VSVG, PRSV, pMDL) são transitoriamente transfectadas em células humanas 293. Depois de pelo menos um período de incubação de 48 horas, o sobrenadante contendo o vírus é colhido e concentrada. Finalmente, título de vírus é determinado pelo repórter (fluorescente) expressão com um citômetro de fluxo.

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

Lentivirus Production

Para fazer lentivírus, vetores de DNA são transfectadas em células humanas 293. Após a colheita e concentrando-se o sobrenadante, título de vírus é determinado pela expressão de fluorescência com um citômetro de fluxo.

Produção lentivírus

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

65.4K visualizações.  University of California, San Francisco - UCSF. Para fazer lentivírus, vetores de DNA são transfectadas em células humanas 293. Após a colheita e concentrando-se o sobrenadante, título de vírus é determinado pela expressão de fluorescência com um citômetro de fluxo.

Vídeo: 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.

Demonstração de técnicas Whole Genome hibridização genômica comparativa de matriz

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 ...

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.

Grandes Inserir Produção Ambiental Biblioteca Genômica

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.

Geração de células-tronco pluripotentes induzidas pela reprogramação fibroblastos humanos com o Lentivirus TF Stemgent Humanos 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.

Um protocolo para a Produção de KLRG1 tetrâmero

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.

Extração de DNA de material parafinado Embedded para análises genéticas e epigenéticas

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.

Transdução de células humanas com Polymer-complexado Lentivirus Ecotropic para reforço de Biossegurança

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.

A Plataforma de Produção de Proteína Complexo MultiBac no EMBL

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

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.

Verde Fluorescente à base de proteínas Expressão Rastreio de Proteínas de Membrana em<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.

Produção de 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 ...

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

Optimal Lentivírus produção e cultura celular condições para sucesso pilhas transdução primária brônquicas humanas epiteliais

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...