Hello guys!! I just have a doubt and I hope someone could give me an explanation. I have been doing lentiviral transduction and lipofection for quite a while. In both cases, absence of serum is found to have increased transduction/transfection efficiency!! May I know how presence of serum affects the efficiency?? Thank you. I hope my question is not very trivial or dumb!!!
You have good answers from a cell biology perspective. I can answer the other side of the story, YOUR CONSTRUCT! Serum, contains albumin which negatively interacts with your lipo-reagent or your virus limiting their ability to interacts with cellular receptors/ membrane.
With Regard to your lipofection, Lipofectamine for example works best when it aggregates upon addition of DNA. This will form big positively charged particles which will sediment onto the cells and fuse/ intracellularly taken by interacting with the negatively charged lipid membrane. BSA contained in the FBS will block two things, the aggregation necessary for your reagent with DNA to sedement and the positive charge on these particles necessary to enter the cells, hindering their efficiency.
To solve this, the easiest way is that you may need to form your lipofectamine-DNA in non-serum containing media, leave for 30min at 37C and add to the serum containing wells, without replacing the media afterwords. The efficiency of transfection in this case will not be severely hindered.
Hope that helped !
Hi Anuroop,
I actually had a research on this question that how serum starvation can increase the transfection efficiency?
What I found as my answer, serum starvation synchronizes cells in G0/G1 stage of the cell cycle. which eventually increases the transfection efficiency.
Hope this would help
Negin
Dear Anuroop and Negin,
G0/G1 proliferation delay/cell cycle cynchronization is one side of the process. The other not less important impact is the rivalry of tranfection agents/viral vector vehicles with big serum proteins for receptors/cell surface approach. Foetal serum proteins are also eager to enter cells in culture, and cells like to get in touch. These two mechanisms are reasons why it is useful to remove serum in advance and perform in vitro transfection/transduction in serum-free medium.
Greetings,
E.F.
You have good answers from a cell biology perspective. I can answer the other side of the story, YOUR CONSTRUCT! Serum, contains albumin which negatively interacts with your lipo-reagent or your virus limiting their ability to interacts with cellular receptors/ membrane.
With Regard to your lipofection, Lipofectamine for example works best when it aggregates upon addition of DNA. This will form big positively charged particles which will sediment onto the cells and fuse/ intracellularly taken by interacting with the negatively charged lipid membrane. BSA contained in the FBS will block two things, the aggregation necessary for your reagent with DNA to sedement and the positive charge on these particles necessary to enter the cells, hindering their efficiency.
To solve this, the easiest way is that you may need to form your lipofectamine-DNA in non-serum containing media, leave for 30min at 37C and add to the serum containing wells, without replacing the media afterwords. The efficiency of transfection in this case will not be severely hindered.
Hope that helped !
Hi Hareth, that was pretty clear and precise. Thank you so much!!
Hi Negin and Elena.. Thank you for the answers. I didn't notice it.. It was actually interesting to get answers from different perspective.! I was actually about to ask, how does it affect in terms of cell biology as I didn't notice your replys. Sorry for that!! Thanks a lot!
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