GFP and siRNA Questions

[p53]

Hi,

 

I searched some publications but found nothing so I decided to ask you here.

 

Did anybody know if it is effectual for a proper flourescence to transfect one single GFP plasmid into an eukaryotic cell?

 

Is it possible for siRNAs to be be exported out of one cell (wich is transfected with shrna vector) into the medium so that other cells (wich are not transfected) can uptake them, so that they also show target protein silencing? Are there described mechanisms for eukaryotic cells for "spontaneous" sirna/mrna or dna export out of the cell or the uptake of siRNA/mRNA or dna?

 

Hope you can help me.

[ecoli]

IIRC, there was an experiment were they inserted the GFP gene in some mammal. The fingernails and hair follicles were flourescent. I don't believe this was done on a plasmid however...

[CharonY]

Regarding GFP, it depends, how do you want to detect the GFP? Moreover, what is the strength of the promoter controlling the gfp gene?

 

Regarding siRNA I have not enough personal experience on this issue. I can only imagine this happening if there is a massive production if the respective siRNA. I can more easily imagine a transfer of the whole vector, though.

[p53]

Regarding GFP, it depends, how do you want to detect the GFP? Moreover, what is the strength of the promoter controlling the gfp gene?

 

Regarding siRNA I have not enough personal experience on this issue. I can only imagine this happening if there is a massive production if the respective siRNA. I can more easily imagine a transfer of the whole vector, though.

 

Hi thanks for your answer. I detected GFP with fluorescence microscopy in vitro and in vivo. I transfeted a psiSTRIKE (shRNA) vector with a gene for hMGFP, which is Monster Green Fluorescent Protein, improved variant of GFP with less cytotocity. Downstream from the hMGFP gene there is an ubiquitary CMV-Promotor.

 

I cotransfected Cos-7 cells with vectors for target protein and psiSTRIKE (shRNA) vectors. My observations shows that cells that doesn't show green fluorescence also have a reduced protein level. So I supposed, that those cells could have uptaken a psiSTRIKE vector that produce shRNAs but not enough hMGFP for a proper fluorescence. Another interpretation would be the export of siRNAs from positive transfected cells into the medium and the uptake by others. But I don't know enough about DNA export and uptake. I know there are core pores in the nucleus membrane that recognize NLS signal from core proteins, and also mRNA export into the cytoplasma, but is there a similar mechanism/transporter/porine for DNA/mRNA export in the cytoplasmic membrane¿ I read in a publication of Elbashir et al. that it is possible for lung cells and different tumor cells to uptake siRNAs without being transfected. So the Cos-7 are kidney cells, immortalized by a tumor process, could this be the answer?

[CharonY]

Did I understand it correctly that you cotransfected cells with a vector carrying the gene for the target protein and a vector with the siRNA sequence, which also carries hmGFP?

 

In that case a simpler explanation might be that there are trans reactions between the promoters of the target protein (or more precisely, gene that codes for the target protein) and that of the hMGFP (I assume you meant that the CMV-promoter is upstream of hmGFP?). This is especially likely if the target gene/protein is also under the control of a strong promoter and enhancer elements. In that case one would expect that they outdilute each other, thus reducing MGFP as well as target protein production.