4 replies to this topic

[s2paik]

Recently I have transducted/transfected cells with a GFP tagged-protein, and observed that there were some noticeable difference
in protein expression (by fluorescence visualization) between the lentivirus transducted and plasmid transfected cells.
The sequence of plasmid used for virus transduction and transfection have been validated using sequencing. Same cell line was used.

Is this difference in expression between virus transducted and plasmid transfected cells expected?
If not, have anyone had any experience with such results, or have any suggestions/comments for this?

Theoretically, shouldn't the protein expression be the same?

Any suggestion, comments are appreciated!!

[bob1]

What sort of difference?  Intensity? distribution? number of cells GFP+?...

[k_undertoe]

When you use a virus, the packaged virus has associated factors that allow it to be integrated and transcribed if it is a lentivirus/retrovirus.  Are you using an adenovirus? A sendai virus?  Depending on what your virus is, it can integrate stably or multiple times into the host genome to give expression at different levels.  The method of transfection/transduction is also dosage sensitive, giving a larger or smaller number of plasmid copies delivered into each cell.

[JoanaLapa]

Hello,

I'm working with astrocytes in mouse models and i would like to do transfection. Do you know what the best reagent to transfect this cells? Lipofectamine or Fugene 6 or other? A colege can order lipofectamine for me, but I'm afraid to buy and then it may not be the best method.  

Thank you!

[bob1]

Any of them should work, but the best one is difficult to say.  For primary cells, I would steer away from lipofectamine 2000, as it tends to be quite toxic.  If you can find one, electroporators are usually the best for primary cells.