So there are a couple things that are confusing in your question, but hopefully I can help. You state you have a lentivirus with a unique promoter for selection. Does this mean that it is a cell type specific promoter, or do you just mean your lentivirus has a selection marker (like neomycin, hygromycin, puromycin, etc) as opposed to promoter? The second question is what protein are you trying to over-express? You state you did a p24 western blot before infection but in early stage of selection... that seems backwards, so I am confused. If you could answer these questions, it would shed some more light on this problem.

Additionally, there are a couple things you should figure out before you start selection. The first is your infection efficiency. You can do this by using a reporter lentivirus (like GFP), and seeing how many cells are GFP+ after infection. If this is low, this could explain why you see little protein after infection. Another thing to do is perform a kill curve for your cell line using the selection agent. Use non-infected cells, and treat different dishes with a range of doses. This will tell you how sensitive the cell line is to the specific drug and how much. This is important because if you find a dose that does not kill the cells, or causes spontaneous resistance, you need to use more or your cells will not be selected correctly. Finally, if you do everything correctly (high infection efficiency, good selection dose, etc) and you still do not have good expression, it could mean a couple things. First, the virus could be bad. Best way to test this is infect and harvest the cells 24-72 hours later and look for your protein (so before selection). If your protein is not highly expressed at that time point, then your virus is not working (could be an array of reasons). The other idea is that sometimes cells actually silence the gene of interest, but not the resistance gene. This is relatively rare, but I have seen it. Some cells can recombine during the selection process... ESPECIALLY if the gene of interest you are over-expressing is has a negative selection pressure on the cells. In this case, only the cells that are expressing the resistance gene, but have low levels of your gene of interest will be selected. This is pretty rare, so try to tackle the others first. Good luck.

~Adam

Thanks Adam! You mentioned that some cells might silence the gene of interest, but not the resistance. We thought about that before, when we were using a lentivirus vector with two promoters. That's what I meant with the unique promoter. It's just one promoter that controls my target gene (it's a viral protein named LANA), and the blasticidin (for selection).

The p24 I did to check how was the virus production before I did the infection. I don't have GFP, that's why I am trying to check my infection rate with IFA for my protein within 3-4 days infection. If the cells don't have the protein and died, it's ok, the problem would be the virus, but around 15-20% of the cells survive, and start growing well.

I have checked the concentration of the antiboitics before starting the tests. My normal cells died in 3 days with 10ng/mL of blasticidin, while my infected/survived ones can go up to 50 and they look fine.

I thought about silencing, but it's too early. And if the virus were bad, I think the cells won't survive.

This is the second lentivirus vector that I am trying, because the first one had 2 promoters. This one is a low copy plasmid, so I am concerned that the quality of DNA is not perfect, but I don't understand how the cells survive.

Right now I am trying to improve the lentivirus protocol.

Thank for all the help!

Suzane

Hi, Suzane.

You told that the WB level of p24 was really low, which means your lentivirus titer was low. So I suggest you to optimize to produce high-titer virus first. If possible, insert a GFP gene in your lentivecter. You may feel troublesome now, but that reporter will save you much time in future.

As for packaging lentivirus, I have some advice.

1. Use two packaging cell lines. Did you use 293T ?

You may try ecopack-293 to produce the first virus and then use it to infect Pt67 to produce the virus you will use for stable cell line generation.

After infection of Pt67, you may harvest the virus supernatant after 3 days. By this time, you may get high-titer amphotropic lentivirus.

2. Incubate the virus producing cells in 32 degree C, this may enhance the production.

3. You may try centrifuging infection. Add virus supernatant into cells you plated, and centrifuge the plate with low speed, like 1000rpm.

As for drug resistance selection, sometimes some cells just survive even they are

supposed to die.

Hope my answer can help.

Wish you lucky.

Guoli Shi

Although your control cells died at 10ng/ml of blasticidin, for efficient selection of cells, you need to use at least 2ug/ml of blasticidin in the culture for efficient selection. Continue the selection procedure for at least a week before you assay for protein expression. Check online for reference levels of blasticidin for the cell culture system you are using to arrive at the concentration in an easier fashion.

Alternatively, you could do a 'kill curve' to determine the effective blasticidin concentration. Briefly, use a range of concentrations of blasticidin to determine the amount of cells surviving post selection, in contrast to the control cells that dont have any selection marker. You may achieve a plateau of concentration at which no more amount of blasticidin can kill any more cells in your culture. That should be the most effective concentration.

Thanks everyone!!