How get GFP signal without mRNA target from pIRES-EGFP - positive by FACS but no DNA amplifation from RT-PCR (May/03/2007 )

I used pIRES-EGFP to get stable clone. This stable clone resists geneticin and give GFP signal detected by FACS. However i couldn't see green cells under fluorescence microscope. Actually I don't have antibody to detect my protein. So i chose RT-PCR to see the expression of my gene. The problem is why i don't have mRNA of my gene examinined by RT-PCR whether this clone is false positive.

very appreciate to get ur opinion

ppapon

---

This is from the pIRES-EGFP description: "pIRES-EGFP contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus
(ECMV) between the MCS and the EGFP (enhanced green fluorescent protein) coding region. This
permits both the gene of interest (cloned into the MCS) and the EGFP gene to be translated from
a single bicistronic mRNA. pIRES-EGFP is designed for the efficient selection (by flow cytometry
or other methods) of transiently transfected mammalian cells expressing EGFP and another protein
of interest. To optimize the selection of cells expressing high levels of the protein of interest,
pIRES-EGFP utilizes a partially disabled IRES sequence (1). This attenuated IRES leads to a
reduced rate of translation initiation at the EGFP start codon relative to that of the cloned gene. This
enables you to select those cells in which the mRNA, and hence the target protein, is produced at
high levels to compensate for a suboptimal rate of translation of EGFP. This vector can also be used
to express EGFP alone or to obtain stably transfected cell lines without time-consuming drug and
clonal selection."

From the description, this vector DO NOT have neo gene, so it CAN'T be resistance to G418.
One more thing, IF you do have GFP expressing cells, it IS possible that you may not see it under microscope since the expression of GFP can be quite low. But I am afraid that the clone you have may not be real.

---

Try extracting DNA and PCR your EGFP-gene. If it is expressed, you should at least have the DNA in your cells. RT-PCR is better for checking expression itself, but working with RNA is more tricky, so I would check presence of DNA first.

---

Thanks for ur responses

Sorry to gave a wrong information, I used pIRES2-EGFP containing Neo/r so firstly i screened the stable clone by antibiotic selection. After i examined survival clones by FACS.

Because I have to write thesis. If it's fake clone, I would like to have any posible reason to explain how it can happen.

Anyway i'm going to try with gDNA as well.

Thanks

---

I'm working with EGFP cells as well (C166-GFP & EOMA-GFP). I can detect it from both FACS and Flu-microscope. It's just tricky to take fluorescence images. You might not be able to see them with the lens. You have to use an image capturing program and adjust to high gain and low shutter speed in order for you to detect them.

Hope this helps.

---

Just a quick question as I don't know the sequence or features of the plasmid: IRES means internal ribosome entry site, so that's not a promoter. This will only function on translational level, meaning that integration of your DNA must have taken place in an intron. So, in case it has integrated elsewhere, you will get geneticin resistant without seeing EGFP, nor seeing it on mRNA level...

Or is there a promoter in the plasmid?

---

Yes, it have CMV promoter in front of the MCS (for insert DNA), then IRES, then EGFP (with Kozak sequence). So the insert and the EGFP will be in one bicistronic mRNA. The insert should not be integrated elsewhere. I am also at a lost here about the RT-PCR. Here are some blind questions: do your insert have the first ATG? Have you sequenced and sure that you have the insert, in frame...? Will your protein be modified (e.g. got signal peptide and be cut off...)?

---

I put cDNA of my gene containing both start and stop codons. DNA sequence was checked after subcloning. it was in flame. I couldn't know whether this protein will be probably modified in PC-3 cells or not. This protein is desaturase which it supposes to modify fatty acid composition in these cells.

ppapon

---

With Kozak sequence? Anyway, it seems that the pIRES2-EGFP may express GFP at low level, so that you cannot see by your eyes under microscope but the FACS can. You can lyse cells, together with untransfected cells, and WB checking for GFP.

About the RT-PCR, afraid that I may not be able to help much since I have never done RT-PCR before. But vairus is right, you can check for your DNA to see if you have your construct in the cells or not first. The problem mostly is because no Ab against your protein. You could add a tag on your protein, I suppose, but most likely you don't like to have tagged proteins that is why you are using IRES. But if worse come to worst, you might need to.

Sorry, don't have any better idea than that. Maybe others more exprerienced could help you?

---