Hello, Andy.

Survived, but no detectable immunoreactive bands. If virus titer is low, producing minimal amount of proteins, they may survive well enough. However, the target proteins is not enough to be detected on blot.

I can only think of Checking your titers of all. Expand viral particles enough.

Best wishes.

Sang Ho: Ok I will try to concentrate the virus and try again. Thanks!

Anand: Thanks. I think the clone and promoter should be fine because the construct express well in 293T cells during transfection.

Hi, Andy.

I just forgot to let you know that pCAG, which we used this promoter in our study of re-cloned of somatic cells. That GFP pig had pCAG-GFP construct. The CAG was coined from pCMV minimal fragment, chicken beta-Actin intron and rabbit beta-Globin fragment (https://en.wikipedia.org/wiki/CAG_promoter)

Regards.

Hi Andy,

Besides Sang Ho's recommendations, I would recommend that you try three things

1- a reporter gene for example GFP, to be cloned the same way as your gene of interest as a control. 2- you can also try to use a reporter construct (another reporter vector to produce a positive ctrl virus) 3- try another cell line

These to rule out a gene transcription/translation issue, viral titer issue or a cell line issue (a possible pre/post translational processing) without including these controls.

Good luck!

Shaimaa's suggestion is a good one - replace your GOI with GFP and see if you can detect that. Pay close attention to how you cloned your GOI, do you have the right Kozak sequence etc?

You may well have very low expression of your GOI - transfection really doesn't mean anything in this context as depending on your MOI, you may have one virus particle per cells vs thousands to millions of plasmids per cell. The difference in expression would be huge.

@Jill

Thanks for your comments!

Yes, I have a GFP plasmid which can be used by lentivirus (But I didn't create that at the same time as my GOI, other member generated it and they confirmed it works). I am now testing if that GFP plasmid work, under the same condition of my GOI. It will tell if that is due to low MOI problem.

If this is not due to low MOI, I might need to re-clone the plasmid again, although I really don't know what is the problem in cloning (Kozak sequence is included and the inserted was sequenced)

Hi Andy,

I did have one more thought: how big is your GOI? Is it very large? If it is, that could cause problems.

@Jill

No it's small, ~700bp (it's a viral protein with ~26kD size)

One information that you have provided regarding the protein, i.e., your GOI is a viral protein. SO, my assumption will be based on that. But before that, I would like to know how many times you have repeated the experiment.

You can use the viral particles to transduce HEK293T again and check if you are getting you protein signal by WB or not. If yes then it has to do something with the cell and some cryptic role of the protein in your target cell line. If you don't get band in WB, then there might be some problem in the construct which might have happened either during transfection in HEK293T during viral particle synthesis or during transduction in your target cell.

So for the later case, you can transduced different cell and see if you are getting the band.

HEK293T has has other viral proteins which makes it a packaging cell line. Its my wild guess that your protein might have some role to play with proliferation and cell cycle.

It could also be that there is gene silencing happening from the cell once the viral DNA integrates into the host genome. Therefore, you're not going to get protein expressed.