Name: Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells (Video) | JoVE Journal
Uploaded: 2007-12-04T04:22:00.000+00:00
Duration: 9 min 40 s
Description: Learn to produce replication-defective retrovirus by transient transfection of 293T cells for modeling oncogene-induced myeloproliferative disease in mice. This method enables safe viral stock preparation.

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<ol>
<li>The evening before transformation, plate 293T cells at 3-5x106 cells/6 cm tissue cultureplate. Note: We use 293T cells for their ease of transfection and efficacyas virus producing cells. These cells are deficient in packaging the virus unless a helper plasmid is introduced. </li>
<li>The first morning, gently remove medium and replace with 4 ml 293T cell med containing 25 mM Chloroquine. Put in incubator for 1 hr. Note: the plate should be about 80% confluent. </li>
<li>Meanwhile, prepare virus-making mixture for 2 plates at a time, in 5 ml tubes:<ol>
<li>2 x 10 mg retroviral plasmid construct</li>
<li>2 x 5 mg packaging construct (EcoPak), </li>
<li>2 x 62 ml CaCl</li>
<li>complete to 1 ml with sterile ddH2O Note: While the retroviral plasmid encodes the oncogene of interest and a marker, EcoPak encodes gag-pol-env. Together with the 293T cells, they will produce an ecotropic retrovirus(RV) specific for mouse cells, but not infective for human cells. Both Chloroquine and CaCl2 have been shown to increase transfection efficiency. </li>
</ol></li>
<li>Add 1ml of 2x sterileHBS drop-wise to the mixture, while gently vortexing the tube.</li>
<li>Immediately add this solution to 2 plates, 1 ml each, gently, drop by drop. </li>
<li>Put in incubator for 7 to 11 hrs.</li>
<li>In the evening (7 to 11 hrs. later), gently remove medium, and very gently replace with 5 ml fresh 293T medium. Put in incubator until noon, the next day. Note: this step is important, as you need to take off the chloroquine from the cells. If kept for extended periods of time, chloroquine is toxic. The solution in the plates looks fuzzy, due to a very fine, dust-like precipitation of the transfection mixture. Note: It is important to do all media changes with extreme gentleness, as the cells have been sensitized by the chloroquine, and can easily detach from the plate. </li>
<li>Next day, 18 to 24 hrs. before retrieving the virus (around noon), gently remove medium, and very gently replace with 3 ml fresh 293T medium. Replace in incubator O/N.</li>
<li>The third morning (18 to 24 hrs. later), gently suck up the medium form the plates with a 10 ml syringe fitted with an 18 G needle. This supernatant contains the retrovirus.</li>
<li>Change the needle to a 45 mm syringe filter, invert syringe several times to mix the solution well, pass supernatant through filter, and aliquot in cryotubes Note: Alternatively, if you are making 3+ plates of virus, pool all supernatants of the same virus into a 50 ml conical tube. Mix well, then aliquot supernatants containing the retrovirus in cryotubes by filtering. </li>
<li>The tubes containing the virus-full supernatants are kept in -80&deg;C until used.</li>
</ol>

<ol><li>DuBridge, R. B., Tang, P., Hsia, H. C., Leong, P. M., Miller, J. H., Calos, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+mutation+in+human+cells+by+using+an+Epstein-Barr+virus+shuttle+system%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.</a> Mol Cell Biol. 7, 379-387 (1987).</li>
<li>Cherry, S. R., Biniszkiewicz, D., van Parijs, L., Baltimore, D., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retroviral+expression+in+embryonic+stem+cells+and+hematopoietic+stem+cells%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Retroviral expression in embryonic stem cells and hematopoietic stem cells.</a> Mol Cell Biol. 20, 7419-7426 (2000).</li>
<li>Finer, M. H., Dull, T. J., Qin, L., Farson, D., Roberts, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((kat%3A+A+high-efficiency+retroviral+transduction+system+for+primary+human+T+lymphocytes%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">kat: A high-efficiency retroviral transduction system for primary human T lymphocytes.</a> Blood. 83, 43-50 (1994).</li>
<li>Naviaux, R. K., Costanzi, E., Haas, M., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pCL+vector+system%3A+rapid+production+of+helper-free%2C+high-titer%2C+recombinant+retroviruses%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses.</a> J Virol. 70, 5701-5705 (1996).</li>
<li>Pear, W. S., Miller, J. P., Xu, L., Pui, J. C., Soffer, B., Quackenbush, R. C., Pendergast, A. M., Bronson, R., Aster, J. C., Scott, M. L., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+and+rapid+induction+of+a+chronic+myelogenous+leukemia-like+myeloproliferative+disease+in+mice+receiving+P210+bcr%2Fabl-transduced+bone+marrow%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow.</a> Blood. 92, 3780-3792 (1998).</li>
<li>Pear, W. S., Nolan, G. P., Scott, M. L., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+high-titer+helper-free+retroviruses+by+transient+transfection%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Production of high-titer helper-free retroviruses by transient transfection.</a> Proc Natl Acad Sci USA. 90, 8392-8396 (1993).</li>
<li>Stocking, C., Kollek, R., Bergholz, U., Ostertag, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long+terminal+repeat+sequences+impart+hematopoietic+transformation+properties+to+the+myeloproliferative+sarcoma+virus%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Long terminal repeat sequences impart hematopoietic transformation properties to the myeloproliferative sarcoma virus.</a> Proc Natl Acad Sci USA. 82, 5746-5750 (1985).</li>
<li>Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Models+of+chronic+myeloid+leukemia%5BTitle%5D)+AND+%22Curr+Oncol+Rep%22%5BJournal%5D)">Models of chronic myeloid leukemia.</a> Curr Oncol Rep. 3, 228-237 (2001).</li>
<li>Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studying+the+pathogenesis+of+BCR-ABL%2B+leukemia+in+mice%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">Studying the pathogenesis of BCR-ABL+ leukemia in mice.</a> Oncogene. 21, 8643-8651 (2002).</li>
</ol>

Four critical points will assure the success of a good viral stock: &nbsp;&nbsp;&nbsp;
<ol>
<li>The 293T producing cells have to be very healthy, meaning they have been split on a very regular schedule, were never overgrown, and are plated at the optimized density of 3.5-5 million per 6 cm tissue culture plate, so that they reach a density of 80% of the plate on the morning of the transfection.&nbsp; </li>
<li>Addition of chloroquine improves transfection efficiency and subsequent virus titer, about 3- ...

<table><tbody><tr><td>293T cells</td><td>cell-line</td><td></td><td></td><td>Split 1:3 to 1:4 every 3 days, otherwise they will tend to clump with replating and will not transfect as well. </td></tr><tr><td>293T cell medium</td><td></td><td></td><td></td><td>DMEM/hi-glu + 10 % FBS + 1% Pen/Strep + 1% L-Glutamine (1% NEAA, optional). We obtain our tissue culture slutions from CellGro.</td></tr><tr><td>coding DNA plasmid</td><td></td><td></td><td></td><td>Twice purified by CsCl. MSCV backbone, encoding oncogene and marker of choice (Neo, GFP, etc.)</td></tr><tr><td>EcoPak</td><td></td><td></td><td></td><td>also called pMCV-Ecopac: ecotropic packaging plasmid, encoding gag-pol-env</td></tr><tr><td>2x HBS</td><td></td><td></td><td></td><td>For 500 ml: 8.0 g NaCl+ 0.37 g KCl + 106.5 mg Na2HPO4 (anhydrous; 201.1 mg if 7xH2O) + 1.0 g dextrose (D-glucose) + 5.0 g HEPES powder. Dissolve in 450 ml dH2O (milli-Q), adjust pH to 7.05 with NaOH, then complete to 500 ml with dH2O. Sterile filter thru 0.45 &amp;#x03BC; filter. Store at room temperature, with the cap on tight.</td></tr><tr><td>2M CaCl2</td><td></td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>miliQ H2O</td><td></td><td></td><td></td><td>sterile-filtered</td></tr><tr><td>Chloroquine</td><td></td><td></td><td></td><td>1000x stock is 25 mM in PBS- (w/o Ca2+/Mg2+), stored at -20C. Add fresh to the medium when needed.</td></tr><tr><td>6 cm plates</td><td></td><td></td><td></td><td>for tissue culture</td></tr><tr><td>Incubator</td><td></td><td></td><td></td><td>for tissue culture. Set at 37C, 10% CO2.</td></tr><tr><td>10 cc sterile syringes</td><td></td><td></td><td></td><td>Sterile. One per virus type.</td></tr><tr><td>18G needles</td><td></td><td></td><td></td><td>single use, one per virus type.</td></tr><tr><td>45 um syringe filters</td><td></td><td></td><td></td><td></td></tr><tr><td>5 ml plastic tubes</td><td></td><td></td><td></td><td>Sterile, to mix transfection solution, for up to 2 plates at a time (2 ml). We use Falcon tubes.</td></tr><tr><td>50 ml conical tubes</td><td></td><td></td><td></td><td></td></tr><tr><td>cryovials</td><td></td><td></td><td></td><td>2 and 4 ml, for virus aliquots.</td></tr><tr><td colspan="5">&lt;p&gt;All reagents used in the transfection must be sterile-filtered (CaCl&lt;sub&gt;2&lt;/sub&gt;, 2x HBS, miliQ H&lt;sub&gt;2&lt;/sub&gt;O) and kept sterile.&lt;/p&gt;</td></tr></tbody></table>

production of replication-defective retrovirus by transient transfection of 293t cells

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

We are here at the Molecular Oncology Research Institute at Tufts New England Medical Center. My name is Christina Lesco and I'm a postdoctoral fellow in Dr.Rick Van's lab. Our lab works on studying human immunogen leukemia, CML and CML like diseases.We are modeling this human disease into mice and the way we're doing it is by introducing the human oncogene through a retroviral vector. So the first thing to do in order to introduce this, this human oncogene into mouse cells is to make the virus itself. The next thing to do will be to actually tighten the virus.So this is my plate of stock, 2 9 3 T cells. I'm gonna have to wash the medium away with PBS 2 9 3 T cells are adherence cells, however, they're not super a adherent, so every movement has to be quite gentle. Once it's washed, we are going to replace the PBS with trypsin.In order to ize the cells out of the plate, we need to place the plate back in the incubator for about three minutes. So after two three minutes, the cells are actually in suspension. We are going to add a few more ml of regular medium.This is going to neutralize the tripsin. At this point, we're populating up and down in order to be sure that all the cells are nicely in suspension, we're passing the suspension to a 50 ml tube and to count the cells in presence of triple blue and use a hemo cytometer to count it by hand. So I'm doing my dilution now as calculated and now I'm going to plate three 10 to the six cells per plate.In four ml medium, we are making sure that the cells are well plated and we are going to put the plates back in the incubator for 16 to 18 hours, which is usually the next morning. So we are now 48 Hours after GFP virus infection. The chloroquine is used in order to increase the uptake of the DNA that we're gonna put on the cells.We gently sucked the media away from the cells, which have now adhere to the plates gently on the side of the plate. At this point, we are going to replace the plates back in the incubators for about one hour. Meanwhile, we are going to prepare our DNA mixture to apply to the cells tier one that is expressing a neomycin resistant gene and the other one that is actually expressing GFPI am using XI DNA in order to control for the entire transfection.Finally, we're adding calcium chloride. The use of calcium chloride greatly increases the transfection efficiency. We are now one hour later, We took about the plates with and chloroquine medium and we are going to add our transfection solution.The last step that we have to do is to actually add one ml of two x sterile HBS while adding the H two XHBS solution. We need to vortex the tube in the same time and go slowly drop by drop. This is to avoid precipitations, immediately distribute one ml of solution to the plate of interest and we are doing that again drop by drop.At this point, the 2 9 3 T cells are quite sensi sensitized. Because of the chloroquine medium that we're having in. We need to have the all the area covered and to move the plates as little as possible, labeled the plate with the construct that you put on and repeat for the next construct.At this point, replace the plates in the incubator for seven to 11 hours. At least seven, but no more than 11 because the cells are very sensitized because of the chloroquine and we will need to Change medium after this time. We are now about 30 Hours after transaction.This is our last change of medium at this point, the 2 9 3 C3 T cells have reached confluence. We have, again, to be very gentle when applying the new medium on the side of the plate. I am using 10 ml syringe with a needle.I'm going to replace the needle with a filter, so I'm mixing the medium well at this point we have collected our virus and we can place it into minus 80 degrees until used before using it. The first thing to do is to actually tighten the virus. There are several sensitive steps while making virus, and the first one of them is the right concentration of 2 9 3 T cells.We are plating them at three 10 to the six cells plate because after 18 hours they will be about 80%confluent. That still leaves them the time to grow more, so therefore 24 hours after transfection they will be 100%confluent. It has been shown that this is quite important for an excellent virus titer.The next step is the use of chloroquine and using chloroquine actually sensitizes the 2 9 3 cells and all the changes of medium have to be done very gently in order for the the entire sheet of 2 9 3 T cells not to lift up from the plate. Finally, the DNA that you're using to make virus has to be extremely clean. We are using C banded, DNA.Once we have our virus, we keep it in minus 80 degrees. We are usually just following it once by each fo will decrease the tighter of the virus two times. Therefore, we like to freeze the virus that we did and therefore forward before we are doing the actual titer of the virus.Depending on the viruses, we may have either GFP expression with the virus to track the actual infected cells, or we can have neomycin resistant. And it is very important to actually have the titer of the virus before using it in order to be able to match between experiments or between different mutants.

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Learn to produce replication-defective retrovirus by transient transfection of 293T cells for modeling oncogene-induced myeloproliferative disease in mice. This method enables safe viral stock preparation.

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

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