Name: transduction of human cells with polymer-complexed ecotropic lentivirus for enhanced biosafety
Uploaded: 2011-07-24T14:00:00
Duration: 14 min 3 s
Description: Watch this Scientific Journal Video about Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety at JoVE.com

La financiación de este proyecto fue proporcionado por el Instituto de Medicina Regenerativa de California.

1. Lentivirus production, harvest, and freezing
<ol>
	<li>Consult your institutional safety official before beginning this protocol, and follow their recommended safety guidelines.</li>
	<li>Day 1. Start with healthy, rapidly growing 293T cells to produce virus. Plate the cells at 5 x 106 
	cells per 10 cm plate. Use antibiotic-free 293T medium (high-glucose DMEM with 10% FBS and 4 mM L-glutamine) for virus production.</li>
	<li>Day 2. In the late afternoon, transfect 293T cells as follows (numbers given are for one 10 cm plate). 
	Let all reagents warm up to room temperature. Pipette 375 &mu;l OptiMEM into a microcentrifuge tube, then add 25 &mu;l 
	Fugene HD. Do not allow undiluted Fugene to contact the surface of the tube.</li>
	<li>In a microcentrifuge tube, mix:
		<ul>
			<li>5 &mu;g transfer plasmid (Slc7a1, target vector, or fluorescent control vector)</li>
			<li>3.75 &mu;g packaging plasmid (pCMV-dR8.91 or psPax2)</li>
			<li>1.25 &mu;g envelope plasmid (pMD2.G for pantropic, pHCMV-EcoEnv for ecotropic)</li>
			<li>serum-free OptiMEM to 100 &mu;l</li>
		</ul>
	</li>
	<li>Combine the two tubes and incubate the mixture 20-30 minutes at room temperature.</li>
	<li>Change the medium on the 293T cells to 10 ml fresh antibiotic-free 293T medium. Add the Fugene/plasmid mix 
	drop-wise to the plate and incubate overnight at 37&deg;C, 5% CO2.</li>
	<li>Day 3. Change medium on the 293T cells to 10 ml fresh antibiotic-free 293T medium. Be gentle, 
	because 293T cells adhere only loosely and can slough off by media pipetted too forcefully onto the 
	monolayer. Incubate for two days in a humidified incubator for virus production.</li>
	<li>Day 5. Harvest virus and filter with a 0.45 mm low protein binding filter. Use immediately or dispense 
	into single-use aliquots and freeze at -80&deg;C. Frozen pantropic and ecotropic virus should be re-titered after it has 
	been stored for six months and one month, respectively.</li>
	<li>Titer the virus as follows, using cells that are relatively amenable to transduction. Transduce cells with fluorescent control virus overnight using serial dilutions (e.g. 1:10, 1:100, 1:1000) in fresh medium with 6 &mu;g/ml PB. Change to fresh medium the next day. Allow the cells two days after transduction to begin expressing the fluorescent protein, then determine the fraction of transduced cells by FACS. Calculate the titer in transforming units (TU) per ml, based on dilutions that yield &lt; 15% transduction to minimize multiple transduction events. 
	<img src="/files/ftp_upload/2822/2822eq1.jpg" alt="Equation 1" />
	</li>
</ol>
2. Transduction of human target cells with murine retrovirus receptor Slc7a1
<ol>
	<li>Select a multiplicity of infection (MOI) = 2 to ensure that most cells will be transduced. For target cells that are resistant to transduction such as HDFs, a higher MOI will be required; we dilute the viral supernatant only 1:2 to achieve transduction of a majority of the cells.</li>
	<li>Transduce target cells overnight with viral supernatant diluted in fresh culture medium with 6 &mu;g/ml PB, which increases transduction by enhancing electrostatic interaction between the virus and target cell.1 Ideally one should use a minimal volume of virus, such as 1 ml for a 35 mm plate, because diffusion is a limiting factor in transduction efficiency. </li>
	<li>Culture cells for at least 48 hours after removing virus before transducing them with ecotropic virus, in order to ensure sufficient expression of the Slc7a1 receptor.</li>
	<li>(Optional) It is possible to use blasticidin to select for stably transduced cells expressing Slc7a1, if desired. A blasticidin kill curve should be generated in advance to determine the lowest effective concentration, generally between 2 &#x2013; 10 &mu;g/ml, that kills all untransduced target cells in 7 days. About 2 days after removing the Slc7a1 virus, switch the transduced cells to medium containing blasticidin. Culture the cells in blasticidin-containing medium for 7 days, at which point all remaining cells will stably express the retrovirus receptor at which point they can be transduced with ecotropic virus as well as banked for future use as a stable Slc7a1-expressing line.</li>
</ol>
3. Polymer complex titration to determine toxicity
<ol>
	<li>Day 1. Plate cells at 5000 cells/well in a 96-well plate, allowing at least triplicate wells for each experimental group. Include untransduced cells in complete medium to provide a baseline value for healthy cells, as well as samples consisting of cells in serum-free medium to induce growth arrest as a control. In parallel, plate cells in an appropriate format (such as a 24-well plate) for microscopic or FACS-based analysis of transduction efficiency in each experimental condition.</li>
	<li>Day 2. Transduce cells in plates set up for both the MTT and FACS analyses with virus encoding GFP, at an MOI of 0.5 to transduce ~40% of target cells. Test varied concentrations of CS/PB (e.g. 50, 100, 200, 400, 600, and 800 mg/ml of each component), as well as 6 &mu;g/ml PB, to determine optimal amounts. Generate polymer complexes as described in Part 4, below.</li>
	<li>Day 3. Remove virus-containing medium and replace with fresh medium. Return the plates to a humidified incubator.</li>
	<li>Day 5. Analyze FACS plate to determine the transduction efficiency with each polymer concentration. </li>
	<li>Day 6. Remove the medium from all wells of the MTT plate and replace with 100 &mu;l MTT solution (1 mg/ml MTT in complete medium). Culture for three hours in a humidified incubator to allow MTT to be reduced in the mitochondria of metabolically active cells.</li>
	<li>Remove MTT solution and replace with 200 ml MTT solvent (0.1 N HCl, 0.1% Igepal CA-630 in isopropanol). </li>
	<li>Incubate for two hours at room temperature or until all of the purple MTT formazan precipitate is dissolved. Read the absorbance on a microplate reader at 570 nm, subtracting the background reading at 690 nm. </li>
	<li>Select the optimal polymer concentration that produces maximum enhancement of transduction, as determined by FACS, with minimal effect on metabolic activity, as determined by MTT. For HDFs, we selected 100 &mu;g/ml CS/PB based on the data shown in the Representative Results, below.</li>
</ol>
4. Ecotropic transduction with polymer complexation
<ol>
	<li>Prepare sterile-filtered stock solutions of PB and chondroitin sulfate at 20 mg/ml in water. Aliquot and store at -20&deg;C.</li>
	<li>Pipette viral supernatant into a microcentrifuge tube and dilute to the desired final concentration (e.g. MOI of 2) with fresh culture medium. Add equal volumes of PB and chondroitin sulfate (concentrations determined in step 3 above) in succession, flicking the tube to mix after each addition. The viral mixture will immediately become cloudy as precipitates form. Incubate the viral mixture at room temperature for 5 minutes to allow complex formation.</li>
	<li>Remove media from the receptor-expressing target cells, replace with the viral mix, and incubate overnight at 37&deg;C, 5% CO2. After removing the viral mix, wash the surface of the cells twice with PBS to aid in removing viral complexes. Complete removal is not necessary; we have observed no adverse effects on cell health from residual polymer complexes.</li>
</ol>
5. Verifying specificity of ecotropic transduction
<ol>
	<li>When producing ecotropic virus for the first time, it is prudent from a safety perspective to verify that the virus is unable to transduce unmodified human cells. Transduce human cells with ecotropic lentivirus at relatively high concentration (only a 1 to 2-fold dilution of viral supernatant in fresh medium) overnight with 6 &mu;g/ml PB or optimized CS/PB concentration. </li>
	<li>Remove virus-containing medium and replace with fresh medium. After incubating cells for 2 days, validate transgene expression by FACS using fluorescent vectors and/or by RT-PCR with transgene-specific primers.</li>
</ol>
6. Representative results:
Fig. 1A shows the lentivirus production process, and Fig. 1B shows transduction of human cells with ecotropic lentivirus, including pre-transduction with murine retrovirus receptor Slc7a1. For titering virus, we use a human rhabdomyosarcoma cell line stably transduced with Slc7a1 (Slc-hRMS). When using fluorescent control vectors to monitor transduction efficiency, we routinely achieve > 90% transduction efficiency of Slc-hRMS with ecotropic virus, as shown in Fig. 2A and 2B. Transduction rates of HDFs are generally lower because the cells have not been blasticidin-selected for receptor expression (Fig. 2C). Titers of ecotropic lentivirus are generally 10-20% of VSV-pseudotyped virus when measured on Slc-hRMS (Fig. 2D).
One freeze-thaw cycle reduces titer of ecotropic virus by 16 - 3%, equal to titer loss during a single freeze-thaw cycle of VSV-pseudotyped virus (p > 0.05). Contrary to previous reports,2 we observe no positive effect on virus titer from flash freezing in dry ice. Rather, we achieve higher post-thaw titers of either pseudotype by simply placing tubes of virus into a -80&deg;C freezer (data not shown). 
The MTT viability assay allows sensitive detection of growth arrest in target cells. Transduction with virus plus concentrations of CS/PB up to 800 &mu;g/ml has no effect on HDF metabolism (Fig. 3A). Exposure to CS/PB without virus also has no toxic effect on cells (data not shown). FACS analysis of HDFs transduced with virus plus various concentrations of CS/PB shows enhancement of transduction compared to PB alone (Fig. 3B). The maximum enhancement occurs at 100 &mu;g/ml CS/PB (Fig. 3C), several-fold lower than previously reported values.3 Thus, it is important to optimize conditions for any given target cell type.
Complexation with CS/PB enhances the observed titer roughly 3-fold in Slc-hRMS (Fig. 4A, p &lt; 0.01). In practice, this yields a greater effect on transduction efficiency at low virus concentrations than at higher concentrations (Fig. 4B), which is most likely due to multiply transduced cells and receptor saturation at higher virus concentrations.
Transduction with ecotropic virus is specific for cells expressing murine retrovirus receptor. When transducing unmodified human cells with ecotropic virus, we have not observed fluorescence greater than the untransduced background whether using PB or CS/PB, as shown in Fig. 5A. Microscopically, HDFs show no transduction in the absence of receptor (Fig. 5B) while pre-transduction with receptor results in fluorescent cells (Fig. 5C).
<img src="/files/ftp_upload/2822/2822fig1.jpg" alt="Figure 1" /> 
Figure 1. Schematic view of (A) the virus production process and (B) transduction of human cells with murine retrovirus receptor followed by ecotropic lentivirus.
<img src="/files/ftp_upload/2822/2822fig2.jpg" alt="Figure 2" /> 
Figure 2. High-efficiency transduction of human cells with ecotropic lentivirus encoding GFP. Cells were pre-transduced with murine retrovirus receptor Slc7a1. (A) Human rhabdomyosarcoma cell line blasticidin-selected for Slc7a1 (Slc-hRMS), transduced (blue) or untransduced (pink) with GFP. (B) Fluorescent micrograph of ecotropic-transduced Slc-hRMS (4X). (C) HDFs transduced (blue) and untransduced (pink) with ecotropic GFP lentivirus; cells were transduced with Slc7a1 two days prior to ecotropic transduction. (D) Titer of VSV-G pseudotyped and ecotropic lentivirus, measured on Slc-hRMS.
<img src="/files/ftp_upload/2822/2822fig3.jpg" alt="Figure 3" /> 
Figure 3. Optimizing chondroitin sulfate/polybrene (CS/PB) concentrations in human dermal fibroblasts. (A) Viability of HDFs measured by MTT assay after transduction in the presence of PB only or with varied concentrations of CS/PB. (B) Enhancement of transduction efficiency in HDFs by different concentrations of CS/PB, measured by FACS. (C) Quantification of transduction enhancement, showing that maximum transduction occurs at 100 &mu;g/ml CS/PB.
<img src="/files/ftp_upload/2822/2822fig4.jpg" alt="Figure 4" /> 
Figure 4. Effect of 400 &mu;g/ml CS/PB on transduction of Slc-hRMS with ecotropic lentivirus. (A) Titer enhancement, and (B) fold change in transduction rate compared to 6 &mu;g/ml polybrene alone.
<img src="/files/ftp_upload/2822/2822fig5.jpg" alt="Figure 5" /> 
Figure 5. Lack of ecotropic transduction of HDFs in the absence of murine retrovirus receptor Slc7a1. (A) FACS plot of BJ human fibroblasts, untransduced (pink) or transduced (blue) with ecotropic GFP lentivirus in both cases in the absence of receptor Slc7a1. BJ human fibroblasts transduced with ecotropic GFP lentivirus, without (B) and with (C) pre-transduction with receptor Slc7a1 (4X).

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Recombinante gammaretrovirus ecotropic basado en el virus de la leucemia murina Moloney (MLV) y su receptor mSlc7a1 están bien estudiadas y ampliamente disponible, después de haber sido utilizado por más de 20 años para ofrecer los transgenes en las células murinas. Gammaretrovirus ecotropic también se ha utilizado más recientemente para ofrecer oncogenes a las células humanas;. En el marco de la reprogramación celular, el uso de mSlc7a1 para evitar la generación de virus anfotrópico albergar oncogenes humanos está bien establecida 4,5 Sin embargo, lentivirus ofrece ventajas significativas sobre gammaretrovirus en la transducción de las poblaciones de células refractarias, 6 incluidas las células primarias que a menudo son objetivos deseables para la reprogramación, ya que el lentiviral pre-integración compleja permite la transducción de la no división de las células 7. Los lentivirus se han producido decenas de pseudotipos diferentes, incluyendo MLV, en un esfuerzo por alterar el tropismo del virus, la toxicidad y otras propiedades. 8-MLV pseudotyped lentivirus ecotropic se ha utilizado para la transducción de células de ratón, 9, pero rara vez se ha utilizado en las células humanas 10. Por tanto, proponemos el uso de MLV pseudotyped lentivirus ecotropic como un vehículo seguro, rentable y altamente eficiente para entregar oncogenes conocidos o sospechosos, incluyendo factores de reprogramación celular, las células humanas. Es importante notar que este protocolo no se elimina totalmente la necesidad de producir y utilizar lentivirus pantropic, sino que este protocolo se separa del oncogén (s) del virus de la pantropic, el aislamiento de los investigadores del potencial de auto-inoculación con virus relacionados con el cáncer. El mSlc7a1 proteína y su homólogo humano hSlc7a1 se expresó doquier transportadores de aminoácidos sin tumorigenicidad conocido o capacidad de conferir una ventaja selectiva de crecimiento de las células receptoras, 11 mSlc7a1 realización de un riesgo relativamente bajo para su incorporación en anfotrópico virus. Este paso adicional puede ser especialmente útil en los laboratorios carecen de virus dedicados o instalaciones de cultivo de tejidos del nivel de bioseguridad necesarias. En algunas situaciones puede ser posible eliminar por completo el uso de virus pantropic mediante la transferencia de la Slc7a1 plásmido directamente en las células diana, sin embargo, muchas células que sería útil para los objetivos de esta técnica también son refractarios a la transfección. Como alternativa, la posibilidad de aislar Slc7a1-células transducidas por la selección blasticidin significa que VSV-G lentivirus pseudotyped puede ser usado una vez para generar una acción de los receptores de las células que expresan, tras lo cual puede ser el virus de ecotropic utilizarse de forma rutinaria para la transducción de estas células para muchos experimentos. Los investigadores siempre deben seguir las directrices de seguridad institucional para trabajar con cualquier lentivirus, independientemente de su tropismo. Los títulos de virus ecotropic logra con este protocolo son ligeramente inferior a la VSV-pseudotyped virus, por lo general el 10-20% de los títulos de virus pantropic, de acuerdo con estudios anteriores. 9 Estos títulos se midieron en las células humanas establemente transducidas con Slc7a1, por lo que el menor título observado para el virus de ecotropic puede deberse en parte a la expresión variadas del gen del receptor en las células diana. Sin embargo, los títulos virales obtenidos en nuestro protocolo es más que suficiente para la mayoría de las aplicaciones y llevar a la transducción de la mayoría de las células, en algunos casos&gt; 90% de las células. Basado en las técnicas de centrifugación se utilizan para concentrar las partículas virales. Sin embargo, la centrifugación requiere varias horas, genera aerosoles infecciosos, y puede resultar en una pérdida significativa de partículas virales. 12 Como alternativa, la formación de complejos con CS / PB se pueden utilizar para mejorar la transducción, sin alterar el tropismo viral. 3 Este método es rápido (5 minutos ) y de bajo costo ($ 0,03 por el virus de 10 ml), mientras que aproximadamente el triple del título observado. Ningún equipo especial o de los reactivos de esta naturaleza se requiere, y se observó toxicidad aguda mínima después de la exposición de HDFs a CS / PB, de acuerdo con trabajos previos en otras líneas celulares. 13 Un posible inconveniente de este protocolo es que algunos de los complejos polímero visibles al microscopio se adhieren a las células durante varios días en la cultura. No podemos descartar la posibilidad de que estos complejos, aunque no visiblemente tóxicas para las células, pueden tener efectos más sutiles en los procesos celulares. Aquí hemos descrito una metodología para la seguridad y eficiencia de transducción de células humanas con factores oncogénicos. Este enfoque debe ser de gran utilidad para los investigadores que estudian los oncogenes y la biología de células madre, incluyendo las células iPS.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> pLenti6/UbC/mSlc7a1 </td><td> Addgene </td><td> 17224 </td><td> Murino receptor MLV </td></tr><tr><td> pMD2.G </td><td> Addgene </td><td> 12259 </td><td> VSV-G sobre </td></tr><tr><td> pCMV-dR8.91 </td><td> D. Trono de laboratorio 14 </td><td></td><td> Embalaje plásmido (plásmido psPax2 equivalente está disponible en Addgene, cat. Número 12.260) </td></tr><tr><td> pHCMV-EcoEnv </td><td> Addgene </td><td> 15802 </td><td> Sobre ecotropic </td></tr><tr><td> FUGW </td><td> Addgene </td><td> 14883 </td><td> GFP plásmido de control </td></tr><tr><td> OptiMEM </td><td> Invitrogen </td><td> 31985 </td><td></td></tr><tr><td> Fugene HD </td><td> Roche </td><td> 04 709 705 001 </td><td></td></tr><tr><td> Hexadimetrina bromuro (Polybrene) </td><td> Sigma-Aldrich </td><td> H9268 </td><td></td></tr><tr><td> El sulfato de condroitina sal sódica del cartílago de tiburón </td><td> Sigma-Aldrich </td><td> C4384 </td><td></td></tr><tr><td> Blasticidin S </td><td> Pescador </td><td> BP 2647100 </td><td></td></tr><tr><td> Methylthiazolyldiphenyl-tetrazolio (MTT) </td><td> Sigma-Aldrich </td><td> M2128 </td><td></td></tr><tr><td> Igepal CA-630 </td><td> Sigma-Aldrich </td><td> I3021 </td><td></td></tr></tbody></table>

transduction of human cells with polymer-complexed ecotropic lentivirus for enhanced biosafety

Madre y el tumor de estudios de la biología celular a menudo requieren la transducción viral de las células humanas con oncogenes conocidos o sospechosos, que planteaba cuestiones importantes de seguridad para el personal de laboratorio. Lentivirus Pantropic, tales como el uso común VSV-G pseudotipo, son una herramienta valiosa para estudiar la función génica, ya que pueden transducir muchos tipos de células, incluyendo las células no se dividen. Sin embargo, los investigadores desean evitar la producción y la centrifugación de los virus pantropic codificación oncogenes debido a los mayores requerimientos de manejo de nivel de bioseguridad y seguridad. Varios oncogenes potentes, incluyendo c-Myc y SV40 antígeno T grande, son conocidos por aumentar la producción de células madre pluripotentes inducidas (IPSC). Todas las otras iPSC que induce cambios genéticos (Oct4, Sox2, Klf4, NANOG, LIN28, y la pérdida de la función de p53) también tienen enlaces con el cáncer, lo que los problemas de seguridad relativamente alto, así. Aunque estos virus relacionados con el cáncer son útiles en el estudio de la reprogramación celular y pluripotencia, deben utilizarse de manera segura. Para abordar estas cuestiones de bioseguridad, se demuestra un método para la transducción de células humanas con lentivirus ecotropic, con énfasis adicional en el costo reducido y fácil manejo. Hemos producido lentivirus ecotropic con título suficientemente alto como para la transducción de más del 90% de los receptores de las células que expresan humanos expuestos al virus, la validación de la eficacia de este enfoque. Lentivirus se concentra a menudo por ultracentrifugación, sin embargo, este proceso dura varias horas y puede producir aerosoles infecciosos para la salud humana de los investigadores biomédicos. Como una alternativa de partículas, viral puede ser más segura sedimentado en las células por la formación de complejos con sulfato de condroitina y polibreno (CS / PB). Esta técnica aumenta el título viral funcional hasta 3 veces en células que expresan establemente receptores retrovirus murino, con el tiempo añadido y el coste insignificante. Transducción de los fibroblastos dérmicos humanos (HDFs) es la máxima mejorada utilizando CS / PB concentraciones aproximadamente cuatro veces menor que el valor óptimo se ha informado de líneas celulares de cáncer, lo que sugiere que la concentración de polímero se debe ajustar para el tipo de célula diana de interés. Por lo tanto, describen el uso de methylthiazolyldiphenyl-tetrazolio (MTT) para el ensayo de toxicidad de polímero en un nuevo tipo de células. Se observa la viabilidad equivalente de HDFs después de la transducción viral utilizando complejos polímero o la dosis estándar de polibreno (PB, 6 mg / ml), lo que indica un mínimo de toxicidad aguda. En este protocolo, se describe el uso de lentivirus ecotropic para la sobreexpresión de los oncogenes en las células humanas, lo que reduce los riesgos de bioseguridad y el aumento de la tasa de transducción. También demostrar el uso de complejos de polímeros para mejorar la transducción, evitando la formación de aerosoles centrifugación de las partículas virales.

Watch this Scientific Journal Video about Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety at JoVE.com

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

Los lentivirus son una valiosa herramienta de investigación para explorar la función del gen, sin embargo, los investigadores desean evitar la producción de la codificación de lentivirus pantropic conocidos o sospechosos oncogenes. Como alternativa, se presenta un protocolo más seguro para el uso de lentivirus ecotropic en las células humanas modificadas para expresar el receptor mSlc7a1 ecotropic.

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 ...

Research

JoVE Journal

Biology

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

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Investigación

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Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

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

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

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A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

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One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

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Imágenes de fluorescencia con un nanómetros Precisión (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...