I'm assuming it's mammalian (or animal) cells you're talking about.

How do you test your expression? If it's a functional test, then maybe the cells aren't processing it properly and don't express a functional/secreted version of the protein.

Cells may have kicked out the part of your gene of interest, but not the resistance gene? I've mentioned this here: http://www.researchgate.net/topic/Cell_Culture/post/Creating_stable_cell_line

I guess there are more possibilities and more info (on what you would have expected and why) would be helpful.

I also faced a similar problem. My plasmid consist of fusion gene of hygromycin and gfp, control by cmv promoter, but i found some of my cells do not express gfp but still survive in the hygromycin. Loosing part of the gene and making the cassette not complete, will the functional protein stil be produced?

To be sure that the ORF of the other (unexpressed) gene is present in your target cells why don't you do a genomic PCR for that gene using transfected & selected cells? If you find the full gene is intact then one can think of the possible reasons.

Dear NicKoli,

I check the expression by western blot. As you said, it seems that cells silence the gene expressio. But question is that how we can prevent this silencing.? Another possibility is inefficient work of signal peptide. Do you know how the efficiency of a given signal peptide can be checked.

Dear Robin,

We checked the gene integration by genomic PCR and it was sharply positive.

Hmm, I don't know, but I can still try to make a few suggestions. So, if I understand correctly you think that somewhere between the integrated DNA and the end protein there is a fault.

If DNA doesn't get transcribed (RT-PCR can check for mRNA), then I suggest trying a different/stronger/more reliable and tested promoter.

If the mRNA doesn't get translated, then I don't know, check with literature if it worked and if there were no internal silencing mechanisms or some fundamental faults with that particular gene expression...

If the synthesised protein doesn't get detected, then it's possible it's all degraded or denatured or whatever, but you have to first convince yourself that your detection method and solubilisation procedures are appropriate (i.e. use a positive control with your antibody). For example, some proteins (GPCRs) can precipitate after boiling in the loading buffer and thus wouldn't get loaded onto a gel. There are a lot more potential pitfalls with that...

Hopefully this was helpful.

Nickolai.

Nickolai and gan jen yang...Same problem with me also..im using adenoviral vector the desire gene tag by two gene GFP & IRES..IRES positive after transfection result also positive for my desire gene but no GFP expressed checked by fluorescent microscopy.

I am not sure that whether my cells silence the production of GFP or not. The gene cassette is like below:

CMV promoter - chimeric intron - Hyg+GFP - pA, if the cell transcribe the hyg resistant gene, it should transcribe along the GFP, but I don't know why they can eliminate the GFP. Moreover, some cells express higher GFP but most of the cells express it in a very low manner, can any1 help me explain it ?

Dear Gan,

If some of your cells expresses GFP , then I think you do not have much problem. It is a generally observed phenomen that after transfection some cells express and other do not express related gene. You just need to select expressing cells by limiting diution! But what's your objective of this transfection? This construct you described are generally used for optimization only!

Dear Sa-fa -jnia,

I read that you have a signal sequence in your wanted protein. Maybe that is the problem. It can be that the SS is not well recognized and as soon the protein is produced but misplaced, it can get degraded. options: Change SS, or add a myristoylation for membrane localization or you can try to inhibit the proteosome to test if degradation is your problem. For my cells, the first time point when I see protein production is 4 hours (for EGFP I can see it at 3 1/2), so you could add the proteosome inhbitor around 2 hours after your transfection. Warning: The proteosome is needed for the cell normal functions, so inhibiting will cause a lot of damage in several systems. I will recommend not to use longer that 3 hours if you want to collect the cells in a fit form. I hope you find the problem,

kind regards, Luisa

Dear Luisa

Thanks for your comments. of course it is not easy to change signal peptide but as you suggested I have to change SS if i can not solve problem. But if SS is not work sufficiently, the protein must be produced in cytoplasm!. I am going to check the cell pelet for my protein by western blot in next days. Luisa, what proteosome inhibitor you have used? Please let me know, I may try for it.

Regards

Dear Luisa and Sa-fa -jnia

If the mRNA is not there - the signal sequence is not going to matter. I strongly suggest you check for that first - pick several different primers, negative control (easy - untransfected cells) and positive control (difficult and you'd have to ask your supervisor for suggestion on that, but very highly recommended) for each set of primers (it can be one control, just amplified with different primers) and do the RT-PCR (I used to do the reverse transcription with random primers). Knowing this may save you a lot of frustration in the future.

Dear Muhammad and Gan

Visualising GFP can sometimes be tricky. Before you say that your cells don't express GFP I suggest you get a positive control. I myself had to use anti-GFP antibodies and secondary antibodies conjugated to green fluorophore to visualise GFP and even then it wasn't dead clear if a cell was expressing GFP or not and a negative control was required for comparison.

Cheers,

Nickolai.

Dear Nickolai,

Your comments seems very helpful. We will ckek with RT

BR

sa-fa

I am currently facing a similar transfection problem. I successfully transfected murine melanoma cells with a lentiviral plasmid 5´LTR-CMV-GOI-SV40-ReportergeneRFP/Selcetionmarker-3´LTR.  

GOI - coding region of my GOI has 350bp and an n-terminal signal sequence and should be secreted.

I selected the reporter gene expressing cells by FACS, checked the insertion of the plasmid by PCR (fwd primer binds CMV promotor region and reverse primer binds SV40 region) and verified the PCR product by sequencing. All this worked out fine. When I wanted to check the expression level of my GOI compared to gapdh and to the non transfected cells, the expression levels of my GOI shows up >10 CTs later compared to gapdh, same levels as my untransfected cells. So I am only detecting the "endogenous" mRNA and not the one of my inserted gene.

So either I have a detection problem of the mRNA (I tried different Primer pairs for sybr green - since I could not detect the mRNA with my own designed primers I even checked with Taqman primers - fail!, also reverse transcription with random primers and oligodTs shows no difference) or there is no mRNA transcribed or immediately degraded... 

Technical support from ABMgoods (company from where I purchased this vector)  explained that the polyA signal is at the end of 3´LTR which also means two different mRNAs are made: one starting from CMV until 3´LTR and the second starting at SV40 until 3´LTR. Since my cells are expressing the reported gene (verified by FACS and fluorescence microscopy) there should not be a problem with the polyA signal...

Maybe you have any suggestion - why I cannot detect the mRNA of my transgene?!

Thanks a lot 

Agnes