I would also press Clontech for more information on what changed in their kit. Like He, I find that pLVX works great with non-proprietary second generation packaging plasmids such as MD2.G and psPAX2. Perhaps the packaging system isn't worth the extra money and hassle.

Hi Cristina, What are the clontech packaging plasmids coding for ? What is the structure of your construct ? Does it contain the U3 region in the 5'LTR or another promoter ? What is the lenght of your vector ? Would you need Rev to increase RNA export ?

Emmanuel

Hi Emmanuel

Regarding packaging plasmids the information of the kit is

"Lenti-X HTX Packaging Mixes are plasmid mixtures that provide the necessary viral packaging components in specific, optimized ratios. When your vector and packaging mix are transfected into Lenti-X 293T cells using the Xfect™ transfection reagents, the packaging mix expresses the Pol (RT & IN), Tat, Rev, and Gag lentiviral proteins, and an envelope that determines the viral particle’s tropism...The packaging mix includes an expression vector for the Tet-Off® transcriptional activator (tTA) and uses Tet transactivation to produce very high expression levels of specific viral proteins" .

Our construct is a pLVX vector. "Lenti-X vectors contain the LTRs, packaging signal (Ψ), Rev response element (RRE), and central polypurine tract/central termination sequence (cPPT/CTS) from HIV-1; and include a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE)."

It should be noted that these plasmids has been successfully used with a previous Clontech kit and that our problems appear when Clontech modified this system to "improve" it.

Thanks for your help

Cristina

We don't use Lenti-XT HTX from invitrogen but routinely produce lenti virus using 3rd generation viral packaging system (pVSV_G from stomatitis virus as the envelope) and 293T_HEK as packaging cells. Since it is probably hard to verify each plasmid in that packaging mix, you can only optimize your transfection technique, such as the purity of the plasmid DNA, the confluency of HEK cells, the collection timing of the supernatan, etc. If you have done all of these, perhaps, you need to change it with different packaging system.

Btw, do you mean that you can not get over expression of your gene of interest? if it is your problem, you can put an external promoter such as EF1a, PGK, CMV in your vector plasmid. Sorry if I don't understand you correctly.

From what you say (packaging plasmids, your own construct), it looks fine and it should work. Are you sure of the serum you're using (tetracycline free) ?

Otherwise you may go back to a simpler 2nd generation system (psPAX2 + pMD2.G) that you can obtain from addgene (http://www.addgene.org/lentiviral/packaging/).

Emmanuel

Thanks for your help

Regarding your comment Eko, the question is that using these plasmids we have previously overexpress the protein of interest with a good efficiency. I agree with Emmanuel that perhaps we can use another system. Actually we are using tetracycline free serum. Furthermore, we have checked the efficiency of HEK transfection with a GFP construct and it was around 95%. Thus, we consider that there is a problem related with virus packaging as suggested by Eko. Furthermore, the packaging system is the component of the kit that has been modified by Clontech. The additional problem is that this kit is very expensive (about 3000 € in Spain) and I consider that a product with this price could not give this kind problems.

The clontech kit is indeed expensive. We only bought the pLVX vector from clontech and combined with pdR8.74 and pMD2.G. It works. What is the difference between the 'old' kit and the 'improved' one? By the way,if clontech can not solve your problem, they should provide a new one or give your money back.

I would also press Clontech for more information on what changed in their kit. Like He, I find that pLVX works great with non-proprietary second generation packaging plasmids such as MD2.G and psPAX2. Perhaps the packaging system isn't worth the extra money and hassle.

Thanks Amanda. In view of your experience and that from Daniel, now I have no doubth about the deficiencies of Clontech system. I'm really interested in your proposal. Could you give me more information about your system and the terms and cost of your services?.

Thanks Amanda

I have contacted Maria Scheel for more information about your services.

Cristina

I have followed the recommendation of Daniel and Emmanuel regarding the use of our pLVX expression plasmids with MD2.G and psPAX2.

Can anyone indicate me the optimal transfection conditions (ratio plvx:MD2.G:psPAX2 and transfection reagent) in HEK cells? We will try with Fugene HD and Fugene 6 but perhaps other liposomes function better.

Thanks

Cristina

Hi Christina,

For 293T cells, the optimal DNA/fugene6 is about 3. It works very well for vector production. For a 10 cm dish, seed 4 millions cells the day before transfection; confluence should be about 50% the day of transfection. Use 8.4µg DNA and 26µL fugene in a total volume of 200µL DMEM (8.4 µg DNA is a mixture of 5.2µg of your vector, 2.3µg psPAX2 and 1µg pMD2.G) / 10 cm plate.

Two hours before transfection, change the medium with anitbiotic free DMEM/10%SVF. Transfect. Wash the day after transfection and harvest the vector 24 hours later. You can keep the supernatant in the fridge and pool it with a second harvest one day later. Bee carefull with the cells that will easily detach from the plastic. Filtrate through 0.45µ durapore membrane. Ultracentrifuge or freeze at -80°C.

Best, Emmanuel

Thanks Emmanuel, we will check your conditions.

Cristina