Working gene-coding plasmid does not produce transcript or protein - (Sep/01/2009 )

Dear all, I hope I'm posting in the right place and forgive my english, please.
I'm having some problem with my BC thesis work.

I'm working with an human acute myelogenous leukemia cell-line (KG-1, premonocyte-like suspension cell): these cells produce the transcript for a gene that is not or very poorly traduced in protein (I never got a positive WB for the protein). When I started I only had RealTime expression data about the gene transcription: mRNA expression was quite high so I thought the protein was produced in my cells.

Since the protein I'm talking about is a membrane receptor, and I want to see if its overexpression down-regulates the signal induced bya particular ligand, I decided to transfect the cells with the gene encoding for the protein. The idea is to overexpress the receptor and stimulate cells with the ligand and see if there is a difference with the wild type cells stimulated with the same ligand.

So I borrowed from another lab a plasmid (pCMV) encoding the gene for the protein I want to express in my cells.

I transfected Hec293 cells with the plasmid and after 4 days I lysed the cells and I found the right band overexpressed in my WB (I ran also the un-transfected cell lysate and got no band on this). So I thought that the plasmid was working well.

Since my cell line is not easy to transfect (I tried Lipofectamine, Dotap, Lipofectamine 2000, TransIT and DMRIE-C and none of this products gave a transfection efficiency higher than 1%) I tried to transfect it with the Amaxa Nucleofector. The positive control plasmid encoding GFP (present in the kit) was extremely well expressed in my cells since after 3-4 days they were 80% green.

So I transfected my cells with the plasmid coding for the protein I wanted to overexpress. The first time I tried, before making a WB, I extracted mRNA from cells (4 days after transfecetion, since the max GFP expression was after 3-4 days) and retrotrascribed to run a RealTime PCR. Unfortunately the transcript levels of wild-type cells and transfeceted cells were identical.

I thought I did something wrong during the transfection so I tried again. This time I lysed the cells (again 4 days after transfecetion) and did a WB using the previously transfected Hec293 (correctly transfected with my plasmid, and producing my protein) as positive control. Nothing again.

As I said when I started the experiment I only had the real time data. Few months later I set-up the WesterBlot apparatus and learned how to run a WB. Last time I blotted Wild-Type KG-1 cells, transfected KG-1 cells and positive control (transfected Hek293): the only positive lane was the Hek293 positive control. So both wild-type and transfected cell don't produce my protein.

I'm doing something wrong, or I'm not considering something, but I don't know what!

Somebody has some good tip?

Thank you and sorry for being so not concise!

E3

-EtramIII-

Good description and you asked the right questions.

I suspect that your transfection isn't working or is very transient. Try titrating the time for expression as it may be that you are missing the peak expression, I would do 24, 48, 72 and 96 hours post transfection harvests.

It may be that the CMV promoter doesn't work in your cell line (though it should), what is the promoter on the GFP plasmid you are using as a test? I would also check that the plasmid is what it is supposed to be, occasionally plasmids will get mutated in the bacteria and stop working. Sequence the gene and the surrounding promoters if possible.

If there is a lot of RNA, but no protein, perhaps the protein is toxic or it is expressed only under certain conditions? There is some evidence that some RNAs are stored by cells for use in emergencies.

-bob1-

Thank you for your kind answer bob1!

bob1 on Sep 7 2009, 03:31 AM said:

I suspect that your transfection isn't working or is very transient. Try titrating the time for expression as it may be that you are missing the peak expression, I would do 24, 48, 72 and 96 hours post transfection harvests.

That will be my first try.

It may be that the CMV promoter doesn't work in your cell line (though it should), what is the promoter on the GFP plasmid you are using as a test?

CMV !!!

I would also check that the plasmid is what it is supposed to be, occasionally plasmids will get mutated in the bacteria and stop working. Sequence the gene and the surrounding promoters if possible.

I thought about sequencing the plasmid, but we are short of money, so I was searching for a cheaper and quicker way out from the problem. If harvesting at different times won't work, I think I'll have the plasmid sequenced or I'll buy a new commercial validated plasmid. According to the money needed and owned...

If there is a lot of RNA, but no protein, perhaps the protein is toxic or it is expressed only under certain conditions? There is some evidence that some RNAs are stored by cells for use in emergencies.

That would be something interesting, but actually I lack any idea about how to verify this: having no clues about which stimulus would induce translation of the mRNA stored in the cell, I don't know how to approach the problem. Let's suppose that in my (untransfected) cell line mRNA is produced and stored but not translated, how would you verify this? The cell-line is myeloid-derived and it reacts to inflammatory stimuli: would you try to induce a translation with different pro- and anti-inflammatory classical stimuli (say LPS or IFN-gamma and IL-4 or IL-13)? Or there is a short-cut?

May it be that some miRNA destroys the mRNA before translation? Is this consistent with the fact that I see a lot of mRNA in my cells?

How many other post-transcriptional regulations capable of translation inhibition exist?

Thank you in advance!
E3

-EtramIII-