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

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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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पुनः संयोजक ecotropic Moloney murine लेकिमिया वायरस (MLV) और उसके रिसेप्टर mSlc7a1 पर आधारित gammaretrovirus और अच्छी तरह से अध्ययन कर रहे हैं व्यापक रूप से उपलब्ध है, 20 से अधिक वर्षों के लिए इस्तेमाल किया गया murine कोशिकाओं को transgenes उद्धार. Ecotropic gammaretrovirus भी अधिक हाल ही में इस्तेमाल किया गया है मानव कोशिकाओं को ओंकोजीन देने, सेलुलर reprogramming की संदर्भ में, mSlc7a1 का उपयोग amphotropic शरण वायरस मानव ओंकोजीन की पीढ़ी से बचने के लिए अच्छी तरह से स्थापित है 4,5 हालांकि, lentivirus gammaretrovirus अधिक महत्वपूर्ण लाभ प्रदान करता है दुर्दम्य सेल आबादी, 6 सहित प्राथमिक कोशिकाओं है कि अक्सर reprogramming के लिए वांछनीय लक्ष्य, transducing क्योंकि lentiviral जटिल पूर्व एकीकरण की अनुमति देता है में गैर विभाजित कोशिकाओं के पारगमन 7. Lentiviruses अलग pseudotypes के दर्जनों MLV सहित, एक करने के लिए वायरस tropism, विषाक्तता, और अन्य गुण 8 ecotropic MLV - pseudotyped lentivirus के लिए माउस कोशिकाओं, 9 transduce इस्तेमाल किया गया है लेकिन शायद ही कभी मानव कोशिकाओं पर प्रयोग किया गया है. बदल प्रयास में, के साथ उत्पादन किया गया है 10 इसलिए हम एक सुरक्षित, लागत प्रभावी, और अत्यधिक कुशल वाहन सेलुलर reprogramming कारकों सहित ज्ञात या संदिग्ध ओंकोजीन, मानव कोशिकाओं को देने के रूप में MLV pseudotyped ecotropic lentivirus के उपयोग का प्रस्ताव है. बल्कि, इस प्रोटोकॉल pantropic वायरस से ओंकोजीन (एस) अलग, कैंसर से संबंधित वायरस के साथ संभावित आत्म टीका से शोधकर्ताओं को इन्सुलेट, यह महत्वपूर्ण है ध्यान दें कि इस प्रोटोकॉल पूरी तरह से pantropic lentivirus उत्पादन और उपयोग करने की आवश्यकता को खत्म नहीं करता है. प्रोटीन mSlc7a1 और उसके मानव homologue hSlc7a1 सर्वत्र कोई ज्ञात tumorigenicity या प्राप्तकर्ता कोशिकाओं पर एक चयनात्मक विकास लाभ प्रदान करने की क्षमता, 11 amphotropic वायरस में शामिल करने के लिए अपेक्षाकृत कम जोखिम बना mSlc7a1 के एमिनो एसिड के साथ ट्रांसपोर्टरों व्यक्त कर रहे हैं. यह जोडी कदम समर्पित आवश्यक जैव सुरक्षा स्तर के वायरस या टिशू कल्चर सुविधाओं की कमी प्रयोगशालाओं में विशेष रूप से उपयोगी हो सकता है. कुछ स्थितियों में यह संभव हो सकता है पूरी तरह से Slc7a1 लक्ष्य कोशिकाओं में सीधे प्लाज्मिड transfecting द्वारा pantropic वायरस के उपयोग को समाप्त कर सकते हैं, तथापि, कई कोशिकाओं है कि इस तकनीक का उपयोगी लक्ष्य होगा भी अभिकर्मक के लिए आग रोक. एक विकल्प के रूप में, को अलग करने की क्षमता blasticidin चयन द्वारा कोशिकाओं Slc7a1 - transduced मतलब है कि VSV जी pseudotyped lentivirus एक बार इस्तेमाल किया जा सकता है रिसेप्टर व्यक्त कोशिकाओं, जिसके बाद ecotropic वायरस नियमित रूप से इस्तेमाल किया जा सकता है कई के लिए इन कोशिकाओं transduce के एक शेयर उत्पन्न प्रयोगों. शोधकर्ताओं ने हमेशा किसी भी lentivirus के साथ काम करने के लिए अपने संस्थागत सुरक्षा निर्देशों का पालन करना चाहिए, अपने tropism की परवाह किए बिना. इस प्रोटोकॉल के साथ हासिल ecotropic वायरस के titers मामूली VSV pseudotyped वायरस, आम तौर पर पिछले अध्ययनों के साथ समझौते में pantropic वायरस टिटर के 10-20%, की तुलना में कम कर रहे हैं इन 9 titers मानव कोशिकाओं में मापा गया stably Slc7a1 के साथ transduced. तो, कम वायरस के लिए मनाया ecotropic टिटर आंशिक रूप से लक्ष्य कोशिकाओं में रिसेप्टर जीन के विभिन्न अभिव्यक्ति के कारण हो सकता है. बहरहाल, वायरल हमारे प्रोटोकॉल में प्राप्त titers सबसे अनुप्रयोगों के लिए पर्याप्त से अधिक कर रहे हैं और कोशिकाओं के बहुमत के पारगमन के लिए कुछ मामलों में, सीसा&gt; कोशिकाओं के 90%. Centrifugation आधारित तकनीकों का सामान्यतः वायरल कणों को ध्यान केंद्रित करने के लिए उपयोग किया जाता है. हालांकि, centrifugation कई घंटे की आवश्यकता है, संक्रामक एयरोसौल्ज़ उत्पन्न, और वायरल कणों के महत्वपूर्ण नुकसान में परिणाम कर सकते हैं के रूप में एक विकल्प है, सीएस / PB साथ complexation. वायरल tropism फेरबदल के बिना 12 से पारगमन को बढ़ाने के लिए इस्तेमाल किया जा सकता है 3 इस विधि तेजी से (5 मिनट. ) और (10 मिलीलीटर वायरस के प्रति 0.03 डॉलर) सस्ती है, जबकि मोटे तौर पर मनाया टिटर tripling. कोई विशेष उपकरणों या स्वामित्व अभिकर्मकों की आवश्यकता होती हैं, और हम HDFs / सीएस पीबी प्रदर्शन के बाद अन्य सेल लाइनों में पिछले काम के साथ समझौते में कम से कम तीव्र विषाक्तता, मनाया 13 इस प्रोटोकॉल के एक संभावित दोष यह है कि कुछ microscopically दिखाई बहुलक परिसरों का पालन. संस्कृति में कई दिनों के लिए कोशिकाओं. हम संभावना है कि इन परिसरों, खुलकर कोशिकाओं को विषाक्त नहीं जबकि, सेलुलर प्रक्रियाओं पर अधिक सूक्ष्म प्रभाव हो सकता है बाहर शासन नहीं कर सकती. यहाँ हम सुरक्षित और कुशलतापूर्वक oncogenic कारकों के साथ मानव कोशिकाओं transducing के लिए एक पद्धति में वर्णित है. ओंकोजीन आईपीएस कोशिकाओं सहित स्टेम कोशिका जीव विज्ञान के अध्ययन के शोधकर्ताओं के लिए महान उपयोगिता के इस दृष्टिकोण होना चाहिए.

<table border="1"><tbody><tr><th> अभिकर्मक के नाम </th><th> कंपनी </th><th> सूचीपत्र संख्या </th><th> टिप्पणियाँ </th></tr><tr><td> pLenti6/UbC/mSlc7a1 </td><td> Addgene </td><td> 17224 </td><td> Murine MLV रिसेप्टर </td></tr><tr><td> pMD2.G </td><td> Addgene </td><td> 12259 </td><td> VSV - जी लिफाफा </td></tr><tr><td> pCMV - dR8.91 </td><td> डी. Trono प्रयोगशाला 14 </td><td></td><td> पैकेजिंग प्लाज्मिड (समकक्ष प्लाज्मिड psPax2 Addgene से उपलब्ध है, बिल्ली 12260 संख्या) </td></tr><tr><td> pHCMV - EcoEnv </td><td> Addgene </td><td> 15802 </td><td> Ecotropic लिफाफा </td></tr><tr><td> FUGW </td><td> Addgene </td><td> 14883 </td><td> GFP प्लाज्मिड नियंत्रण </td></tr><tr><td> OptiMEM </td><td> Invitrogen </td><td> 31985 </td><td></td></tr><tr><td> Fugene एच.डी. </td><td> रॉश </td><td> 04 705 001 709 </td><td></td></tr><tr><td> Hexadimethrine ब्रोमाइड (Polybrene) </td><td> सिग्मा Aldrich </td><td> H9268 </td><td></td></tr><tr><td> Chondroitin सल्फेट शार्क उपास्थि से सोडियम नमक </td><td> सिग्मा Aldrich </td><td> C4384 </td><td></td></tr><tr><td> Blasticidin एस </td><td> मछुआ </td><td> 2647100 बी.पी. </td><td></td></tr><tr><td> Methylthiazolyldiphenyl tetrazolium ब्रोमाइड (MTT) </td><td> सिग्मा Aldrich </td><td> M2128 </td><td></td></tr><tr><td> Igepal सीए - 630 </td><td> सिग्मा Aldrich </td><td> I3021 </td><td></td></tr></tbody></table>

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.

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 जीन समारोह की खोज के लिए एक मूल्यवान अनुसंधान उपकरण हैं, हालांकि, शोधकर्ताओं pantropic lentivirus एन्कोडिंग में जाना जाता है या संदिग्ध ओंकोजीन के उत्पादन से बचने के लिए इच्छा हो सकती है. एक विकल्प के रूप में, हम ecotropic lentivirus ecotropic रिसेप्टर mSlc7a1 व्यक्त संशोधित मानव कोशिकाओं पर प्रयोग के लिए एक सुरक्षित प्रोटोकॉल उपस्थित थे.

बढ़ी जैवसुरक्षा के लिए पॉलिमर complexed Ecotropic lentivirus के साथ मानव कोशिकाओं के पारगमन

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

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