Hi Elia,

besides checking the frame/orientation of your insert and mutations in the vector, you might also require to optimise the transfection depending on the plasmid size. How big is the protein you want to express with the GFP-tag?

By optimization I mean changing the DNA:lipofectamine ratio or even changing the cell type. How long after transfection are you checking the cells? Some cell types shut down promoters like CMV after 48h. I was trying to transfect mouse kidney cells with a big vector with no luck, and when I changed to HEK293 cells it worked great straight away.

Is there a chance that your protein might be actively degraded? Ubiquitation, nonsense mediated decay, etc?

Maybe the protein overexpression is negatively selecting those cells. Do you see more dead cells in the culture?

Good luck!

Claudio

Hi Elia,

You don't mention which vector you are using. Make sure your gene insert is in frame with the GFP. If the insert is not in frame your PCR will look fine but the plasmid will be making junk RNA. Also check the sequence to make sure no stop site was introduced during cloning.

Hi Elia,

besides checking the frame/orientation of your insert and mutations in the vector, you might also require to optimise the transfection depending on the plasmid size. How big is the protein you want to express with the GFP-tag?

By optimization I mean changing the DNA:lipofectamine ratio or even changing the cell type. How long after transfection are you checking the cells? Some cell types shut down promoters like CMV after 48h. I was trying to transfect mouse kidney cells with a big vector with no luck, and when I changed to HEK293 cells it worked great straight away.

Is there a chance that your protein might be actively degraded? Ubiquitation, nonsense mediated decay, etc?

Maybe the protein overexpression is negatively selecting those cells. Do you see more dead cells in the culture?

Good luck!

Claudio

Hello everyone and thanks for your prompt replies.

First Daniel, I'm using a pEGFP-N1 plasmid as my vector where I insert a 1.1 Kb gene in the MCS(multiple cloning site). I am sure about the frame/sequence this is why it's so confusing. Second, Claudio, earlier studies have been done in our group on these cells and I'm kind of stuck with them, and I tried changing the ratio without luck. 24 hours post transfection, a lot of dead cells are expected due to the method itself. 48 hours post that I start my antibiotic treatment (for which I already did a kill curve). As for fate for that protein I'm not sure, literature is not very clear on the topic but this has been transfected successfully using different methods though ! The weird thing is that these cells behave differently in culture, which perplexes me, with respect to the empty vector control and the untransfected control.

Elia,

If the transfection efficiency of your modified vector is similar to your empty vector (90%) you would not expect to see too much killing when under selection. After more thought on your problem I realized I had this problem with one of my genes of interest. Do you know how toxic this protein is to your cells? If the gene is highly toxic when over expressed the cells may have turned it off via epigenetic mechanisms. One way to check for this is to treat with Sodium butyrate (inhibits HDAC activity) and see if this results in GFP expression. If the cells turn green you will know that your transfection and construct worked. The bad news is you will have to use switch to an inducible expression vector .

Hi Elia 

what is excitation /emission use for your vector pEGFP-N1 for capturing GFP fluorsecence.Is it 480/520 blue/green? and which was the cell line you have transfected, was it a glial cell line?.

Hi, 

I am not sure if the problem still persists. If you have just checked for fluorescence, don't you also not want to check by western blot if the protein is getting expressed, but GFP is not folding properly? Or by PCR of cDNA made from RNA isolated from the transfectants?

Hi!

I am Having the same probled using HeLa cells with the same plasmid.

Did you manage to make the GFP express in the plasmid containing the insert?