Sandler Asma Centro de Investigación Básica (SABRE) Sandler Fundación

Parte 1: La transfección de células HEK 293 células para producir lentivirus * 24 horas antes de la transfección, la placa de 2,5 X 10 6 células en una placa de 10 cm para una confluencia del 50-70% al día siguiente. <ol><li> Inicio este protocolo mediante la preparación de ADN con la que uno más tarde transfectar una de las células para hacer virus. En primer lugar, diluir FuGENE 6 Transfección reactivo Roche, un reactivo de los lípidos que componen las células toman el ADN. En un tubo de 1,5 ml, pipeta de 30μl FuGENE 6 en 600ul medio libre de suero (SFDMEM). Tenga cuidado de no dejar que FuGENE tocar la parte del tubo que se introduce en el medio. Deja que este FuGENE y mezclar SFDMEM e incubar a temperatura ambiente durante 5 minutos. </li><li> En la tapa del tubo, mezclar 4 mg lentiviral plásmido con 4 mg cada 3 ª generación vectores virales embalaje (pMDL, PRSV y pVSV-G). El lentiviral plásmido contiene el ADN del virus se inserta en el genoma de cada célula que infecta, mientras que los vectores de envases contienen genes de todas las otras proteínas necesarias para hacer un lentivirus. </li><li> Cierre la tapa y mezclar invirtiendo el tubo varias veces. </li><li> Incubar durante 15-20 minutos a temperatura ambiente. </li><li> Pipeta de todo el contenido del tubo en la placa 293. Mezclar suavemente por la placa de inclinación hacia atrás y adelante. </li><li> Volver a las células a la incubadora y permiten la producción viral que continúe durante 48-96 horas antes de la cosecha. </li><li> Durante la incubación, las células 293 transcribir el ADN de interés de la lentiviral plásmido, creando una copia de ARN de la construcción de lentiviral. También se transcriben y traducen los genes en los vectores de envases en las proteínas virales. Estas proteínas se procederá a realizar los virus de ARN que contiene la versión de su construcción lentiviral, que el virus se invertirá transcribir e insertar en los genomas de todas las células que infecta. </li></ol> Parte 2: Cosecha lentivirales <ol><li> Después de un período de incubación, tomar células de la incubadora y el uso de una jeringa desechable, de 10 ml para separar el sobrenadante, que ahora contiene el virus. Habrá alrededor de 10 ml de sobrenadante. </li><li> Coloque un filtro de jeringa 0.45μM a la punta y filtrar los virus en un tubo de ultracentrífuga Beckman. El filtro sólo permite que los virus, y no a las células a pasar. </li><li> Antes de desechar, se incuban todas las células produzcan más virus, placas de cultivo, las jeringas y los filtros en el 10% de lejía durante 45 minutos. </li></ol> Parte 3: Concentración lentivirales a través de ultracentrifugación <ol><li> Carga tubos de centrifugación en SW-41Ti o SW-28 cubos del rotor. </li><li> El uso de suero libre de DMEM para equilibrar todos los virus que contienen los tubos dentro de 0.2g. </li><li> Se cierre cubos del rotor y engancharlos en el rotor antes de colocar el rotor dentro de la ultracentrífuga </li><li> Girar el virus durante 90 minutos en una ultracentrífuga a 4 ° C a 25.000 rpm en un rotor SW-41Ti o 24.000 rpm en un rotor SW-28. </li><li> En una campana de cultivo de tejidos, invierta los tubos boca abajo para verter el sobrenadante en un recipiente con lejía al 10%. </li><li> Use un Kimwipe para absorber el exceso de líquido alrededor de la bolita y se invierte el tubo en un estante conveniente para no más de cinco minutos. </li><li> Para volver a suspender el pellet viral, utilice un filtro de punta a añadir 100 ml de PBS estéril en el tubo, entonces la pipeta hacia arriba y abajo unas 20 veces. </li><li> Deje el virus a 4 ° C durante la noche para completar la re-suspensión. Uno puede almacenar prepara viral a 4 grados durante aproximadamente 1 semana y a -80 grados durante un máximo de un año. Recuerde que debe tratar a todos los tubos de vacío con lejía al 10% antes de desecharlos. </li><li> Si se piensa en usar la preparación viral por más de la semana 1, hacer alícuotas mínimas de 5-20μl para futuros experimentos y una alícuota de 3 a 5μl de la titulación. Congelar y almacenar las alícuotas a menos 80. </li></ol> Parte 4: Valoración lentivirales mediante citometría de flujo <ol><li> El siguiente paso es determinar el título viral por citometría de flujo. Este método sólo funciona en los casos en que los lentivirus plásmido contiene un gen fluorescente. La idea es que cuando el virus infecta una célula, se introduce el ADN del plásmido lentiviral en el genoma de la célula. La célula infectada luego transcribir y traducir los genes que se incluyen en el plásmido lentiviral, y si incluye un gen fluorescente, fluorescente las células infectadas se. El nivel de fluorescencia representa la potencia, o título, de los virus. </li><li> Para cada cepa viral que se titularon, diluir 3 l de virus concentrado en 1,5 ml de medio completo (DMEM, pen-estreptocócica, glutamina, FBS). </li><li> En los pozos de 6.1 en una sola fila de una placa de 96 pocillos, realizar las diluciones seriadas siguiente: a los 6 pozos de añadir 100 ml de DMEM completo los medios de comunicación. Para el primer pozo de añadir 100μl de los medios de comunicación, sin virus (esta es su control sin manchas), para el segundo y añadir 100 ml del virus diluido (lo que equivale a 1 ml de virus sin diluir), a la tercera y añadir 100 mlde la mezcla de la segunda y, a la cuarta y añadir 100 ml de la tercera y, a la quinta y añadir 100μl del pozo cuarto y el sexto y añadir 100 ml de la quinta también. Finalmente tome 100 ul de la sexta y así descartar en lejía. Una serie de dilución se realizó en el que cada pozo tiene la mitad del volumen de los virus en comparación con el anterior. </li><li> Obtener un volumen total en todos los pozos de 200μl. Tenga en cuenta que se trata de diluciones sólo sugirió, que debería funcionar bien para la titulación de los virus más concentrada. Si el virus es baja en el título, o sin concentrar una posible necesidad de ajustar las diluciones. </li><li> Tratar cualquier virus diluido no utilizado con lejía al 10% durante 45 minutos antes de desecharlos en un contenedor de residuos biopeligrosos. </li><li> Añadir ~ 15000 sano, las células se dividen activamente 293 a cada uno de los seis pozos. Una sola placa de 96 pocillos se puede utilizar para títulos de 16 preparaciones virales. </li><li> Volver a las células incubadora grado 37 con 5% de CO 2 y que la infección de proceder por lo menos durante 48 horas. </li><li> Después de la incubación, las células son analizadas para la expresión reportero (en nuestro caso la fluorescencia) con un citómetro de flujo. Las partículas virales se cuantifica el porcentaje de células fluorescentes por pozo. </li></ol> Calcular el número de unidades infecciosas por ml con la siguiente fórmula: <div style="text-align:center;"> (Número de células por pocillo en el día de las células de la infección por el reportero X positivo) X 1000 ______________________________________________________ ml de vector añadido a bien </div> Parte 5: Resultados del representante: Después de un después de la transfección de 48 horas el período de incubación, la placa de 293 células debería ser al fluorescentes alrededor del 90%. Un alto porcentaje de células fluorescentes es una indicación de un alto porcentaje de virus productores de células. Después de la centrifugación, el pellet viral es apenas visible cuando la resuspensión. Esto es normal ya que la pastilla es clara y muy pequeños. Al interpretar los resultados del citómetro de flujo, tenga en cuenta que sólo se puede lograr cálculos exactos título cuando se infectan (fluorescentes), las células no han recibido más de una integración viral por célula. Con el fin de asegurar que este es el caso, utilizar las células con una tasa de &lt;15% de infección para los cálculos. Las células con las tasas de infección más altos probablemente han recibido varios integrantes virales por célula. Las diluciones que se sugiere en el paso 2 debe rendir varias muestras dentro de este rango de infección. Lo ideal es utilizar varias muestras para generar un gráfico de titulación (lineal) de la cual se puede calcular el título final, pero se puede calcular el título con una sola muestra, si es necesario. Uno puede esperar título de 1,0 x 10 7 TU / ml de concentrado sin virus y 1.0 x 10 8 TU / ml de virus concentrados.

<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

ARN de interferencia (ARNi) es un sistema de silenciamiento de genes en las células vivas. El ARN de interferencia, en la secuencia de genes homólogos a corto RNAs de interferencia (siRNA) son silenciadas en el estado post-transcripcional. ARN de horquilla corta, los precursores de siRNA, se puede expresar mediante lentivirus, lo que permite el ARNi en una variedad de tipos de células. Los lentivirus, como el virus de inmunodeficiencia humana, son capaces de infectar tanto a la división y que no se dividen las células. Vamos a describir un procedimiento que a los lentivirus paquete. Envase se refiere a la preparación de virus competentes de vectores de ADN. Lentiviral los sistemas de producción de vectores se basan en un &#39;split&#39; del sistema, donde ha sido el genoma del virus natural de división en las construcciones de plásmido auxiliar individual. Esta división de los diferentes elementos virales en cuatro vectores separados disminuye el riesgo de crear un virus capaz de replicarse por recombinación accidental del genoma del lentivirus. Aquí, un vector que contiene la shRNA de interés y tres vectores de empaquetamiento (p-VSVG, PRSV, pMDL) son transfectadas transitoriamente en 293 células humanas. Después de al menos un período de incubación de 48 horas, el sobrenadante que contiene el virus se recoge y se concentra. Finalmente, título del virus se determina por el reportero (fluorescentes), expresión con un citómetro de flujo.

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

Lentivirus Production

Para que los lentivirus, los vectores de ADN se transfectadas en células humanas 293. Después de la cosecha y la concentración del sobrenadante, título del virus se determina mediante la expresión de fluorescencia con un citómetro de flujo.

Lentivirus de producción

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 vistas.  University of California, San Francisco - UCSF. Para que los lentivirus, los vectores de ADN se transfectadas en células humanas 293. Después de la cosecha y la concentración del sobrenadante, título del virus se determina mediante la expresión de fluorescencia con un citómetro de flujo.

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.

Demostración de técnicas de hibridación del genoma entero matriz Genómica Comparativa

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 ...

Investigación

Biología

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.

Inserte grandes Ambiental Genómica Producción 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.

Generación de células madre pluripotentes inducidas por reprogramación de fibroblastos humanos con los Lentivirus Stemgent Humanos 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 protocolo para la producción de 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.

Extracción de ADN a partir de material incluido en parafina para análisis genéticos y epigenéticos

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.

Transducción de las células humanas con polímeros complejos Lentivirus ecotropic de Mayor Seguridad de la Biotecnología

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.

El Protein Complex Plataforma de Producción MultiBac en el 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.

La producción de embriones de pez cebra mediante haploide<em&gt; In Vitro</em&gt; Fertilización

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.

Fluorescente verde a base de proteína Expresión Proyección de proteínas de membrana en<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.

Producción de ADN de doble cadena 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 ...