Dear Agnes,

I think (and agree with Rocco) that the most likely explanation for the failure of you GOI expression is the silencing of the CMV promoter in melanoma cells. CMV promoter is know to be very weak active in a number of cell lines. You could check if this is the case by infecting with this same construct a cell line which is known (from the literature) to support the CMV promoter driven gene expression. In case, you will still not see the GOI expression, I suggest that you stop troubleshooting and generate another vector in which the GOI will be controlled by another promoter, such as EF1alpha, Ubiquitin C or CAG. In selecting the most promising promoter, you could check the literature whether it is known which promoter is most active in melanoma cells. If you will be constructing another vector anyway, you can than incorporate other improvements suggested in answers above (2A was better than IRES in our hands in polycistronic vectors). If you will still get silencing by using lentiviruses that integrate more or less randomly into genomic areas that may facilitate signaling, you could also consider gene targeting into "safe harbor" locus (ROSA26 in murine cells) using zinc finger nucleases (ZFN) or TALEN technologies. This approach resulted in several fold lesser silencing of transgene expression in our system (murine pluripotent stem cells). Paper describing these data will be published soon in PLOS One (after September 16th; Lepperhof V. et al.) and you are welcome to look at it. Good luck with your project!

You're right, if things went according to plan, this "shouldn't" happen.  Do you know for sure that the CMV promoter is fine?  (did you sequence it? / or restriction digest?). Perhaps something went wrong during cloning.  Sometimes some bp adjacent to your gene can get ruined, which might just affect the transcription start site if you get unlucky.  This would affect only a single (rare) E. coli clone.  Do you have plasmids from other clones that you could try?

Alternatively, could there be a regulatory region hidden inside your "GOI" that's interfering?  (a promoter firing against the CMV, or a hairpin that might terminate transcription before your RT primer?)  This would be really weird to happen in 350 bp.  Most likely a cloning error I would guess. 

Does your GOI have miRNA binding sequence?

sometimes retroviral single copy insertions, could have very low  expressed transcript levels, which may not be too much evident if its a very strongly expressed GOI. Do you have any cell culture conditions where you can reduce the endogenous GOI expression to minimum, to see whether you have any exogenous transcript at all.

It could be a problem of the vector construct. It has been reported (see e.g. http://www.ncbi.nlm.nih.gov/pubmed/16697259)  that promoter interferences can occur between two adjacent promoters (in your case the SV40 and the CMV promoter). These promoter interferences can lead to mosaic expression, no expression or only partial expression (only one of the genes).

Therefore you could switch to a construct with two identical promoters (SV40 and SV40) or to a vector construct harboring an IRES or an self-cleaving peptide (e.g. 2A peptide).

Good luck

Hello Agnes,

CMV promoter can be silentiated through methylation. This often occurs quite fast in presence of a selective pressure.

Could it be that your goi is toxic/detrimental for your cells?

If not, switching to an IRES, as suggested by Tobias, could be a good solution.

1. I think you should be able to choose the pair of primers which could pick up your construct, but not the endogenous protein.

2. I wouldn't  spend my time on investigating problems with current construct because it would not lead me to the aim of my research. As long as making a construct takes just 3 days I would try different system of expression. There are plenty to choose from on the market.

I know people do use expression cassettes in this orientation with success, but I always worry about how well the first gene is getting polyadenylated if the transcript also has to go through a second gene.

If you may be having problems with your PCR, can you check for expression of your GOI any other way (FACS, Western, functional assay etc)?

I agree with the suggestions of using a 2A or IRES (although they can have their own problems....).  We have also had success using a bidirectional promoter (i.e. genes reading away from each other ) from Axel Schambach's lab (Maetzig et al, Gene Ther 2010).

Lastly, is the N terminal signal sequence endogenous or have you added it on?  We have found in some situations that adding these to a gene to try and get the protein secreted can kill the functionality of the protein.

Good luck!

Thanks a lot for all your answers and suggestions!

@ John Schloendorn – I bought the vector completly ready for transfection and sequenced it (pure plasmid before the transfection and PCR product of the plasmid after transfection) which was fine. I bought the same plasmid with three different GOIs (always small proteins, chemokines that shouls be secreted) and I find none of them with PCR. How would I find out out if there is a regulatory hidden region inside my gene?

@Michael B LoMonaco: I don´t know – how can I find this out?

@ Manoj Balakrishna Menon: I bought the same plasmid with three different GOIs, a mouse GOI and two human homologs of it – there should be no basal expression of the human version and in any case I find the GOI with RT-PCR.

@Tobias May: Thanks for your suggestion. if I dont get it work in the next two weeks (finding the GOI expression with PCR or western, which I am now trying, I will have to switch to a different construct and start again with tranfection).

@Rocco Pizza: Methylation is a ggod point, but would methylation occur in the whole transfected cell population? I don't think that my GOI is toxic (small secreted chemokines)

@Steven J Howe: The Nterminal sequence is endogenous – the protein should be secreted. I am trying now western of concentrated cell culture supernatants.

In case, I really have to use a different construct, you would suggest a promotor (CMV, SV-40 – any preferences) the GOI followed by IRES and reporter gene, right?!

Hi Agnes,

you sorted your cells by FACS, so I assume it's no longer the whole population, but only the subset with the highest expression. BTW: what's the efficiency of your infection? Is it possible for you to test the effect of the chemokine on your cells? Could it be that your GOI, even if not toxic, slows down cell growth, induces differentiation or similar ?

There is a data set of miRNA and their targets.  Google will get you there.  Also try GeneCards at Weizman.  Good luck

Besides the ongoing suggestions, have you consider the possibility of some 'hidden' splicing event emerged by way of appearance of some SD/SA site? Even though your GOI is under the regulation of CMV towards the 5' end, and yet upstream LTR may be active (?). Also, is there antibiotic selection? That may again cause the lost of the region of interest.  I will suggest using simpler constructs, and use of bi-cistronic vector using IRES.  Also, given the type of target cells (murine melanoma cells) involved that are dividing cells, simple MuLV based vector pseudotyped with VSVG should do the task (rather then using complex lentiviral vector system with additional sequences).

Dear Agnes,

I think (and agree with Rocco) that the most likely explanation for the failure of you GOI expression is the silencing of the CMV promoter in melanoma cells. CMV promoter is know to be very weak active in a number of cell lines. You could check if this is the case by infecting with this same construct a cell line which is known (from the literature) to support the CMV promoter driven gene expression. In case, you will still not see the GOI expression, I suggest that you stop troubleshooting and generate another vector in which the GOI will be controlled by another promoter, such as EF1alpha, Ubiquitin C or CAG. In selecting the most promising promoter, you could check the literature whether it is known which promoter is most active in melanoma cells. If you will be constructing another vector anyway, you can than incorporate other improvements suggested in answers above (2A was better than IRES in our hands in polycistronic vectors). If you will still get silencing by using lentiviruses that integrate more or less randomly into genomic areas that may facilitate signaling, you could also consider gene targeting into "safe harbor" locus (ROSA26 in murine cells) using zinc finger nucleases (ZFN) or TALEN technologies. This approach resulted in several fold lesser silencing of transgene expression in our system (murine pluripotent stem cells). Paper describing these data will be published soon in PLOS One (after September 16th; Lepperhof V. et al.) and you are welcome to look at it. Good luck with your project!

Thanks Tomo for your help - just a final question: When I go for the EF1a promoter, does it matter if I use the human EF1a for my murine melanoma cells or is this the wrong decision? All the publication I have read about my problem only talk about either the human EF1a promotor or dont mention the species of the promoter sequence at all when they used it in other cell lines than human such as B16.

Sorry, Agnes, for not replying sooner to your question regarding the EF1a promoter. We have been working with human EF1a promoter and we have used it in human cells successfully. Unfortunately, we did not test it in murine cells but I guess that human EF1a promoter would also be active in murine cells.