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

Finansieringen för detta projekt från California Institute för regenerativ medicin.

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).

<ol>
	<li>Davis, H. E., Rosinski, M., Morgan, J. R., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charged+polymers+modulate+retrovirus+transduction+via+membrane+charge+neutralization+and+virus+aggregation.">Charged polymers modulate retrovirus transduction via membrane charge neutralization and virus aggregation.</a> Biophys J. 86, 1234-1242 (2004).</li><li>Marino, M. P., Luce, M. J., 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=Small-+to+large-scale+production+of+lentivirus+vectors.">Small- to large-scale production of lentivirus vectors.</a> Methods Mol Biol. 229, 43-55 (2003).</li><li>Landazuri, N., LeDoux, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexation+of+retroviruses+with+charged+polymers+enhances+gene+transfer+by+increasing+the+rate+that+viruses+are+delivered+to+cells.">Complexation of retroviruses with charged polymers enhances gene transfer by increasing the rate that viruses are delivered to cells.</a> J Gene Med. 6, 1304-1319 (2004).</li><li>Ohnuki, M., Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+characterization+of+human+induced+pluripotent+stem+cells.">Generation and characterization of human induced pluripotent stem cells.</a> Curr Protoc Stem Cell Biol. 4, 4A.2-4A.2 (2009).</li><li>Hotta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EOS+lentiviral+vector+selection+system+for+human+induced+pluripotent+stem+cells.">EOS lentiviral vector selection system for human induced pluripotent stem cells.</a> Nat Protoc. 4, 1828-1844 (2009).</li><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=Transduction+of+nondividing+cells+using+pseudotyped+defective+high-titer+HIV+type+1+particles.">Transduction of nondividing cells using pseudotyped defective high-titer HIV type 1 particles.</a> Proc Natl Acad Sci U S A. 93, 15266-15271 (1996).</li><li>Cullen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Journey+to+the+center+of+the+cell.">Journey to the center of the cell.</a> Cell. 105, 697-700 (2001).</li><li>Cronin, J., Zhang, X. Y., 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=Altering+the+tropism+of+lentiviral+vectors+through+pseudotyping.">Altering the tropism of lentiviral vectors through pseudotyping.</a> Curr Gene Ther. 5, 387-398 (2005).</li><li>Schambach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vectors+pseudotyped+with+murine+ecotropic+envelope:+increased+biosafety+and+convenience+in+preclinical+research.">Lentiviral vectors pseudotyped with murine ecotropic envelope: increased biosafety and convenience in preclinical research.</a> Exp Hematol. 34, 588-592 (2006).</li><li>Koch, P., Siemen, H., Biegler, A., Itskovitz-Eldor, J., Brustle, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+of+human+embryonic+stem+cells+by+ecotropic+retroviral+vectors.">Transduction of human embryonic stem cells by ecotropic retroviral vectors.</a> Nucleic Acids Res. 34, e120-e120 (2006).</li><li>Yoshimoto, T., Yoshimoto, E., Meruelo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+and+characterization+of+a+novel+human+gene+homologous+to+the+murine+ecotropic+retroviral+receptor.">Molecular cloning and characterization of a novel human gene homologous to the murine ecotropic retroviral receptor.</a> Virology. 185, 10-17 (1991).</li><li>Cepko, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+preparation+and+concentration+of+retrovirus+stocks.">Large-scale preparation and concentration of retrovirus stocks.</a> Curr Protoc Mol Biol. Chapter 9, Unit 9.12-Unit 9.12 (2001).</li><li>Doux, J. M. L. e., Landazuri, N., Yarmush, M. L., Morgan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexation+of+retrovirus+with+cationic+and+anionic+polymers+increases+the+efficiency+of+gene+transfer.">Complexation of retrovirus with cationic and anionic polymers increases the efficiency of gene transfer.</a> Hum Gene Ther. 12, 1611-1621 (2001).</li><li>Zufferey, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-inactivating+lentivirus+vector+for+safe+and+efficient+in+vivo+gene+delivery.">Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.</a> J Virol. 72, 9873-9880 (1998).</li></ol>

Rekombinant ecotropic gammaretrovirus baserat på Moloney murina leukemi virus (MLV) och dess receptor mSlc7a1 är väl studerat och allmänt tillgängliga, har använts i över 20 år att leverera transgener mot murina celler. Ecotropic gammaretrovirus har också använts på senare tid för att leverera onkogener till mänskliga celler,. I samband med cellulär omprogrammering, användning av mSlc7a1 undvika generation amphotropic virus hysa mänskligt onkogener är väl etablerad 4,5 ger dock lentivirus betydande fördelar jämfört med gammaretrovirus i transducing refraktär cellpopulationer, 6 inklusive primära celler som ofta är önskvärt mål för omprogrammering, eftersom Lentiviral före integrationen komplex kan transduktion av icke-delande celler. 7 Lentiviruses har producerats med dussintals olika pseudotypes, bland MLV, i ett försök att ändra virus tropism, toxicitet och andra egenskaper. 8 MLV-pseudotyped ecotropic lentivirus har använts för att transduce musceller, 9 men har sällan använts på mänskliga celler . 10 Vi föreslår därför att använda MLV-pseudotyped ecotropic lentivirus som en säker, kostnadseffektiv och mycket effektiva fordon för att leverera kända eller misstänkta onkogener, inbegripet cellplattor omprogrammering faktorer, till mänskliga celler. Det är viktigt att notera att detta protokoll inte helt eliminera behovet av att producera och använda pantropic lentivirus, utan skiljer detta protokoll onkogen (s) från pantropic viruset, isolerande forskare från potentiella själv inokulering med cancer-relaterade virus. Proteinet mSlc7a1 och dess mänskliga homolog hSlc7a1 är ubiquitously uttrycks aminosyra transportörer utan känd tumorigenicitetsprov eller förmåga att ge en selektiv tillväxt fördel mottagaren celler, 11 vilket gör mSlc7a1 relativt låg risk för inbyggnad i amphotropic virus. Denna extra steg kan vara särskilt användbart i laboratorier som saknar dedikerad virus eller vävnad anläggningar kultur krävs biosäkerhetsnivån. I vissa situationer kan det vara möjligt att helt eliminera användningen av pantropic virus genom transfecting den Slc7a1 plasmiden direkt i målceller, men många celler som skulle vara bra måltavlor för denna teknik är också refraktära mot transfektion. Som ett alternativ, förmågan att isolera Slc7a1-transduced celler genom blasticidin val innebär att VSV-G pseudotyped lentivirus kan användas en gång för att generera ett lager av receptor-innehållande celler, varefter ecotropic virus kan användas rutinmässigt för att transduce dessa celler för många experiment. Forskare bör alltid följa sina institutionella riktlinjer för säkerheten för att arbeta med någon lentivirus, oavsett dess tropism. Titrar av ecotropic virus uppnås med detta protokoll är något lägre än VSV-pseudotyped virus, i allmänhet 10-20% av pantropic viruset titer, i samförstånd med tidigare studier. 9 Dessa titrar mättes i mänskliga celler stabilt transduced med Slc7a1, så den lägre observerade titer för ecotropic virus kan delvis bero på olika uttryck av receptorn genen i målceller. Trots den virala titrar uppnås i våra protokoll är mer än tillräckligt för de flesta applikationer och leda till transduktion för majoriteten av celler, i vissa fall&gt; 90% av celler. Centrifugering-tekniker används ofta för att koncentrera viruspartiklar. Kräver dock centrifugering flera timmar, genererar smittsamma aerosoler, och kan resultera i betydande förlust av viruspartiklar. 12 Som ett alternativ, komplexbildning med CS / PB kan användas för att förbättra transduktion utan att ändra viral tropism. 3 Denna metod är snabb (5 minuter ) och billig ($ 0,03 per 10 ml-virus), medan ungefär tredubblas de observerade titer. Ingen speciell utrustning eller egenutvecklade reagenser som krävs, och vi observerade minimal akut toxicitet efter exponering av HDFS CS / PB, i samförstånd med tidigare arbete i andra cellinjer. 13 En potentiell nackdel med detta protokoll är att vissa mikroskopiskt synliga polymer komplex följa cellerna i flera dagar i kulturen. Vi kan inte utesluta att dessa komplex, men inte direkt toxiska för cellerna, kan ha mer subtila effekter på cellulära processer. Här har vi beskrivit en metod för säkert och effektivt transducing mänskliga celler med onkogena faktorer. Denna strategi bör vara till stor nytta för forskare att studera onkogener och stamcellsbiologi inklusive iPS-celler.

<table border="1"><tbody><tr><th> Reagensets namn </th><th> Företag </th><th> Katalognummer </th><th> Kommentarer </th></tr><tr><td> pLenti6/UbC/mSlc7a1 </td><td> Addgene </td><td> 17.224 </td><td> Murina MLV-receptorn </td></tr><tr><td> pMD2.G </td><td> Addgene </td><td> 12.259 </td><td> VSV-G kuvert </td></tr><tr><td> pCMV-dR8.91 </td><td> D. Trönö lab 14 </td><td></td><td> Förpackning plasmid (motsvarande plasmiden psPax2 finns från Addgene, Cat. Number 12.260) </td></tr><tr><td> pHCMV-EcoEnv </td><td> Addgene </td><td> 15.802 </td><td> Ecotropic kuvert </td></tr><tr><td> FUGW </td><td> Addgene </td><td> 14.883 </td><td> GFP kontroll plasmid </td></tr><tr><td> OptiMEM </td><td> Invitrogen </td><td> 31.985 </td><td></td></tr><tr><td> Fugene HD </td><td> Roche </td><td> 04 709 705 001 </td><td></td></tr><tr><td> Hexadimethrine bromid (Polybrene) </td><td> Sigma-Aldrich </td><td> H9268 </td><td></td></tr><tr><td> Kondroitinsulfat natriumsalt från hajbrosk </td><td> Sigma-Aldrich </td><td> C4384 </td><td></td></tr><tr><td> Blasticidin S </td><td> Fisher </td><td> BP 2647100 </td><td></td></tr><tr><td> Methylthiazolyldiphenyl-tetrazolium bromid (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

Stamceller och tumör cellbiologi studier kräver ofta viralt transduktion av mänskliga celler med känd eller misstänkt onkogener, höja viktiga säkerhetsfrågor för laboratorie-personal. Pantropic lentiviruses, som vanligen används VSV-G pseudotype, är ett värdefullt verktyg för att studera geners funktion eftersom de kan transduce många celltyper, inklusive icke-delande celler. Däremot kan forskarna vill undvika produktion och centrifugering av pantropic virus kodning onkogener på grund av högre biosäkerhet krav nivå hantering och säkerhetsfrågor. Flera potenta onkogener, inklusive c-Myc och SV40 stora T-antigen, är känd för att öka produktionen av inducerade pluripotenta stamceller (IPSC). Alla andra kända IPSC-inducerande genetiska förändringar (OCT4, SOX2, KLF4, NANOG, LIN28 och p53 förlust av funktion) har också länkar till cancer, vilket gör dem relativt hög säkerhet oro också. Även om dessa cancer-relaterade virus är användbara för att studera cellulära omprogrammering och pluripotens, måste de användas säkert. För att lösa dessa biosäkerhet frågor, visar vi en metod för transduktion av mänskliga celler med ecotropic lentivirus, med extra betoning på minskade kostnader och bekväm hantering. Vi har tagit fram ecotropic lentivirus med tillräckligt hög titer till transduce mer än 90% av receptor-uttrycka mänskliga celler utsätts för viruset, validera effekten av denna strategi. Lentivirus ofta koncentreras genom ultracentrifugering, men tar detta flera timmar och kan producera aerosoler smittsamt för människor biomedicinska forskare. Som ett alternativ viruspartiklar kan vara mer säkert sjunkit ned cellerna genom komplexbildning med kondroitinsulfat och polybrene (CS / PB). Denna teknik ökar den funktionella virus titer upp till 3-faldig i celler stabilt uttrycka murina retrovirus-receptorn, med försumbar lagt tid och kostnad. Transduktion av mänskliga dermal fibroblaster (HDFS) är maximalt förstärkt med CS / PB koncentrationer ca 4-faldigt lägre än det optimala värdet som tidigare rapporterats för cancercellinjer, vilket tyder på att polymer koncentrationen bör titreras till målcellen typ av intresse. Vi beskriver därför användning av methylthiazolyldiphenyl-tetrazolium bromid (MTT) för analys för polymer toxicitet i en ny celltyp. Vi iakttar motsvarande livskraft HDFS efter virus transduktion med antingen polymer komplexbildning eller standard dos polybrene (PB, 6 mikrogram / ml), vilket tyder på minimal akut toxicitet. I detta protokoll, beskriver vi användning av ecotropic lentivirus för överuttryck av onkogener i mänskliga celler, vilket minskar biosäkerhet riskerna och öka transduktion takt. Vi visar också användningen av polymera komplexbildning att förbättra transduktion och samtidigt undvika aerosol-bildande centrifugering av viruspartiklar.

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

Lentiviruses är ett värdefullt forskningsverktyg för att undersöka geners funktion, men kan forskare vill undvika produktion av pantropic kända lentivirus kodning eller misstänkt onkogener. Som ett alternativ, presenterar vi en säkrare protokoll för användning av ecotropic lentivirus på mänskliga celler modifierats för att uttrycka ecotropic receptorn mSlc7a1.

Transduktion av mänskliga celler med polymer-komplexbundet Ecotropic lentivirus för förbättrade biosäkerhet

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

Transfecting mänskliga neurala stamceller med Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Detection of Protein Ubiquitination

Detektion av protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Generering av inducerade pluripotenta stamceller genom att programmera om musen Embryonala fibroblasts med en fyra transkriptionsfaktor, Doxycyklin inducerbara Lentiviral Transduction System

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

Lentivirus Production

Lentivirus Produktion

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

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

Generering av inducerade pluripotenta stamceller genom att programmera humana med Stemgent mänskliga 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 ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Differentiering av embryonala stamceller till Oligodendrocyte Prekursorer

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Proteomic Provberedning från formalinfixerade och paraffininbäddade vävnads

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

Effektiv Generation Human inducerade pluripotenta stamceller från mänskliga somatiska celler med Sendai-virus

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 ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

Zink-finger Nuclease Enhanced Gene Targeting i mänskliga embryonala stamcellslinjer

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

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence Imaging with One-nanometer Accuracy FIONA

Fluorescens Imaging med One-nanometer Noggrannhet (FIONA)

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