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Problem in transfection for producing lentivirus


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#1 Mindologist

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Posted 02 January 2013 - 10:20 PM

Hi all enthusiast,

I'm looking for good advices about producing lentivirus.
My problem is that there is no RFP (reporter) cells after transfection with viral construct and packaging plasmids.
I'm using second generation packaging system, pPAX and pMDG, and my viral construct is pSIN4 vecter with a gene of my interest.
For producing virual, I'm using 293T cell, and I'm using Calcium phosphate method for transfection.
When I transfect only my viral construct (containing RFP gene), I could see RFP+ cells next day (efficiency is good).
However, after transfection with my viral construct + packaging plasmids, there's no RFP+ cells next day.
I don't know what's going on.

So, I'm looking for senior's advices and solutions.
Thanks a lot for your helps.
Happy new year!

#2 bob1

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Posted 15 January 2013 - 11:37 AM

As it is a multiple plasmid transfection you need to be very careful with the ratios of plasmids (to each other) that you are using and the total amount of DNA you are trying to get into the cell.

I would recommend looking at a different system of transfection other than Ca phosphate, as this is quite inefficient. PEI is cheap (I think about $200 for enough to make 1 liter of solution, and you would use a few ul per transfection) and works well.

#3 Mindologist

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Posted 16 January 2013 - 12:36 PM

I just saw your great comments. Thank you a lot, bob1. I'm going to try different ratio of the plasmids. Do you have any ratio that you may suggest me? If you do, it would be great for me.

Besides, other question is about serum free medium. I searched internet and saw some are suggesting using serum free medium during viral production, but others just use serum-containing medium.
Which one do you think is better?

I really appreciate to you for the good suggestion and sharing knowledge.

#4 bob1

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Posted 17 January 2013 - 12:10 AM

I wouldn't go above about 2 ug total, the ratio will depend a bit on the plasmids - I would start off with 1:1:1, then try ratios up to 1:3 of each.

I use serum free for the transfection, and do the transfection mix in a minimal volume so as to increase the chances of the different DNAs all being in the same complex (I don't think this applies to Ca phosphate transfection though), I then incubate for 1-3 hours in SFM and then add an equal volume of 20% FCS containing medium for the assay duration.

#5 Mindologist

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Posted 18 January 2013 - 07:25 AM

I think that using serum free first and then adding 20% FCS is great idea. Thanks a lot, bob1.




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