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lentivirus production issues


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#1 whitelight

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Posted 27 October 2013 - 05:29 AM

Hi all,

 

I've long been lurking around these forums and often found topics to be really helpful for my own experiments.

 

However, I'm now experiencing some difficulties with lentivirus propagation and any input would be appreciated.

 

A few weeks ago, I did virus preps with 3x 15-cm plates per construct and unfortunately got titers around 10^6-10^7 (concentrated and pellets resuspended in 300ul media).

This is obviously way too low. I did not save any unconcentrated supernatant, hence I don't know whether viruses were lost during concentration, or simply wasn't there to begin with.

 

At the moment, I'm doing some more preps and in short my protocol is: Seed 293T cells (9*10^6 cells) in 15-cm plates the day before transfection and then transfect the appropriate vectors using Lipofectamine LTX.

My plan is to collect supernatants at 48h and 72h post transfection, pool, ultracentrifugate and compare titer with some non-concentrated supernatant. I checked 24h post transfection in a fluorescence microscope and found that pretty much all my cells were green - yay smile.png

 

However, when I harvested the 48h supernatant I found that a substantial number of the packaging cells had dettached and where floating. Is this normal? Cells do seem to have a funny morphological appearance which I hope is due to VSV-G overexpression and syncytia formation. The Lipofectamine LTX manual states that you do NOT need to Exchange the medium post tranfection and I therefore skipped it..

 

Any input would be greatly appreciated. If needed I will update the thread when I know more about the titers in the current experiment.

 

Thanks again.

 



#2 2xzwei

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Posted 21 November 2013 - 08:50 AM

Sometimes virus production/packaging is tricky and behaves strangely without any obvious reason. So repeating the whole process may lead to success (I had that several times, too).

 

293T cells are a bit temperature sensitive, so pre-warm all media before putting it on the cells otherwise they'll detach. During virus production it is normal that some cells detach, die and float around.

 

I never tried lipofection for virus production, I stick to calcium chloride method as it’s cheap, easy and gives good results. Your cell number seems a bit high to me (but I don't use 15cm dishes) - cells should be around 70-80% of confluence. Do not let them overgrow, this will hinder transfection! I start with 1x10^6 cells in 10cm dishes (in the evening), change medium the next morning, do transfection, change medium after 12 hours and then collect supernatant 48h later (medium has not to be changed, it can turn yellow). With half of the supernatant I do ultracentrifugation and freeze a stock, the other half I directly use for transduction which works out for the most part…


Edited by 2xzwei, 21 November 2013 - 08:50 AM.





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