I've always had a lot of difficulty with fibroblast transfection.  I would recommend trying with a mouse neuronal cell line such as Neuro2a or if possible, trying with primary mixed cultures.

Out of curiosity, what tumor type are you trying to induce?  I'm also trying to use lentivirus to induce a brain tumor!  

Thanks Levi, I am working on GBM 

Are you using polybrene when you transduce the 3T3 cells? What sort of an MOI are you using?

Yeah I am  (4 microgram/ml), I have used 2-3 different batches of packaged viruses with different MOI but still same results (MOI 5-8). 

You may need to go higher on the MOI. Like 10-20 or even 40 MOI. Depends on what sort of efficiency you need. I have had to go that high with rat cells before to get >50% efficiency.

Okay I will try doing that. Is it possible that I don't have well formed viral particles and I get only transfer vector transduction or If I try changing packing plasmid from pMD2.G to VSV-G envelop plasmid ?

If you have good transduction efficiency in the HEK cells then you have well formed infectious particles. The pMD2.G plasmid IS the VSV-G envelop protein.

My theory of why trying to transduce rodent cells with VSV-G enveloped lentivirus is difficult (and I haven't investigated this although I will at some point) because they have very low expression of the LDL receptor on their cell surface which is the receptor the VSV-G envelop protein binds to. So if the expression of the LDL receptor is low there is not much you can do except throw a lot of virus at the cells to try and increase the efficiency. On the other hand, human cells transduce well with relatively low MOIs. The other option is to change envelop proteins to one in which its receptor is expressed at a higher level on rodent cells. Unfortunately I don't know what receptor that would be.

Good Luck!

Thanks Tom and everyone

hi

if you easily got  the other cell line transduced, but that one, it means that cell line is not that easy to work on. for example RPE are very hard. they need a lot higher titre of virus. If your insert is not very big you should have already got the expression and if you are just transducing only GFP alone, size of vector is not an issue. Changing the vector may help. pNLEGFPCMVWPREdU3 is very efficient as long as your insert is not bigger than 1 kb. this vector produce particles that almost transduce all kinds of cells. 

if you like to keep working on your vector, I recommend you seed target cells in a 24-well plate and use the virus collected and concentrated from two 10 cm plate and keep it on the cells for 2 hours.

Thanks Mostafa 

Hi Ravinder,

What promoter do you used? We currently transduced 3T3 with VSV LV PGK or EF1 promoters without problem.

Maybe have a look here to optimize your condition of transduction

http://www.geg-tech.com/knowledge/protocols/transduction-with-lenti-one

Regards

Thanks Nicholas I will go through the link, I have U6 and H1 promoters in plasmid

I don't really understand, U6 and H1 are PolII promoters which are used to express RNA and you told that you look GFP expression. What is the promoter which drives the GFP?

Sorry its CMV 

Ok, I think you could try to optimize your conditions of transduction and if it doesn't work,, test other promoters

I will do that thanks

Hi, hope you've got it working by now.

Also, make sure your cells aren't too confluent - I probably wouldn't go above 70%.

Can you get infection with the control virus? A non-silencing with GFP?

Hi Ravinder, are you still struggling with this lentivirus infections? Actually I am having just the same problem. My HEKs are highly infected (>75%) however 3T3 cells are none. Can you help me with it?

Can you please give some details about your lentivirus ? I still have issues but 3T3s are largely sorted out.

yes sure Ravinder. I am using pGFP-C-shLenti, pCMV-VSV-G and pCMV-dR8.2 dvpr to make the virus. I concentrated the virus 100 times through centrifugation and the used 10ul of it to infect 3t3 cells in 12 well plate. they didn't turn out green after 48 hours. later on I used 50ul of to infect 293 cells (in 12 well plate) and they were very green. I am planning to try infection in 3t3 cells with more virus but saw this question so wanted to get some advice before starting the experiment.

Try using higher MOI 50-100, they are very hard to transduce, Use polybrene 5-8microgram/ml.

Spin the plates 30-60 minutes after transduction.