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Troubleshootting lentiviral transduction.


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#1 Wek

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Posted 08 March 2015 - 07:43 PM

I have been trying to transduce a few murine cell lines with a with a lentivirus for the past month and I just dont know where could be the problem. My lentivirus is IRES-GFP so I know that my transfection of 293T cells is good (GFP+ cells after 24 hours). Here's the protocol I follow:

 

Day 0: plate 2e5 293T on a 6 well plate.

 

Day 1: add 1ug or lentivirus, 1ug of each packaging plasmids along with Fugene HD with a 3:1 ratio (9ul of Fugene). I transfect my 293T in 1ml of media complete with antibiotics.

 

Day 2: (24hrs after) add 2ml of media for a total of 3ml.

 

Day 3 (48hrs after) collect the 3ml of old media with virus and add 2ml of fresh complete mdia. Put the old meida in 4C.

 

Day 4: Collect the 2ml of media and pool with old media, filter through a .45um filter and add to my cell lines.

 

I add 1ml of the media containing virus and 8ug/ml to my cells lines (5e4 cells/well on 24 well plate). Spin infect at 37C for 99mins. The next day I see no GFP.

 

Any help would be appreciated.

Thanks

 

 

 

 

 

 



#2 5280

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Posted 11 March 2015 - 02:57 PM

I would optimize your transfer (lenti), packaging and envelope plasmid ratios. They may not be ideal for virus production. You have not listed which packaging vectors you are using for production. Maybe they are commercial and the company should have established this ratio that is ideal. Otherwise you will need to optimize. I use use my transfer, psPAX2 (packaging), pMD2G (env) at the following ratios - 4:3:1. Also, spin transduction is usually not necessary for lentivirus, it is used more commonly for retrovirus.  I usually get pretty high titers at 48hrs and do not add media back for a second collection at 72rs. I collect the media at 48hrs, spin then filter with 0.45u and lastly aliquot this supernatant for freezing at -80C. Also, did you determine that 8ug/ml polybrene is ideal or did you pick that based off literature or it being the highest concentration people use for transduction? It may not be ideal for your cells. 



#3 Rsm

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Posted 13 March 2015 - 03:15 AM

In my hands, mouse cells are difficult to infect with lentivirus. I would suggest to use a human cell line as a control to see if your virus is there. 8ug/ml Polybrene is a bit high, but shouldn't be a big problem (as long as your cells don't die). You may also want to consider up-scaling, I usually use a 10cm dish of 293 for production of virus.


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#4 5280

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Posted 13 March 2015 - 06:39 AM

I agree, I usually produce virus from a 10cm or 15cm. 

 

 

In my hands, mouse cells are difficult to infect with lentivirus. I would suggest to use a human cell line as a control to see if your virus is there. 8ug/ml Polybrene is a bit high, but shouldn't be a big problem (as long as your cells don't die). You may also want to consider up-scaling, I usually use a 10cm dish of 293 for production of virus.






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