Hi,
I was just wondering if anyone has any personal experience or opinions on using PEI for the production of lentivirus in 293T cells compared to calcium phosphate precipitation and lipophectamine.
From what I am aware, calcium phosphate results is very low, while the ever improving reagents such as lipofectamine are quite high (as high as 80% if not higher) but expensive as well !
Any suggestion or comment in this regard will be appreciated.
best
niloufar
Hi Niloufar,
In our lab, we routinely use calcium phosphate for transfection of HEK293T cells for virus packaging, and get comparable transfection efficiency to Lipofectamine. Granted, calcium phosphate still requires an element of magic when preparing the solutions (but its soooo much cheaper!!). In my personal experience the points I found most important were:
- pH of the solution. This is critical. When making the stock of phosphate solution, we make it at several different pH's (in increments of 0.05, from around 6.9 to 7.1) and then check their relative transfection efficiencies. We freeze down the best one and toss the rest.
- quality of DNA. Keep your plasmid stock at a high concentration, in water, and as much of it as possible in a supercoiled state (i.e. purify your plasmid when it has reached the stationary stage, do the purification at low temperature, etc.).
- Cell state: I've seen some people be a bit haphazard with their splitting and allowed their cells get a bit too confluent (albeit a few passages prior) to transfection, and it always turns out to be a lower efficiency. Passage your cells consistently, plate 24 hours before transfection, and make sure you have the appropriate confluence when transfecting.
We have also tried PEI, with good transfection efficiency; however, the PEI-DNA complex is a bit toxic to cells, so you have to be careful about establishing the conditions.
Hi Niloufar,
In our lab, we routinely use calcium phosphate for transfection of HEK293T cells for virus packaging, and get comparable transfection efficiency to Lipofectamine. Granted, calcium phosphate still requires an element of magic when preparing the solutions (but its soooo much cheaper!!). In my personal experience the points I found most important were:
- pH of the solution. This is critical. When making the stock of phosphate solution, we make it at several different pH's (in increments of 0.05, from around 6.9 to 7.1) and then check their relative transfection efficiencies. We freeze down the best one and toss the rest.
- quality of DNA. Keep your plasmid stock at a high concentration, in water, and as much of it as possible in a supercoiled state (i.e. purify your plasmid when it has reached the stationary stage, do the purification at low temperature, etc.).
- Cell state: I've seen some people be a bit haphazard with their splitting and allowed their cells get a bit too confluent (albeit a few passages prior) to transfection, and it always turns out to be a lower efficiency. Passage your cells consistently, plate 24 hours before transfection, and make sure you have the appropriate confluence when transfecting.
We have also tried PEI, with good transfection efficiency; however, the PEI-DNA complex is a bit toxic to cells, so you have to be careful about establishing the conditions.
Dear Kelly ,
Thank you so much for your answer.
Would you let me know your exact protocol for LV production in HEK293Tcells by CaHpo4 please?
Kind regards
Niloufar
@KellyKawabata
Dear Nur,
Thank u so much for your answer.
Best
Nilou
@Nurcokaturk
I have used both, PEI and Lipo based delivery. PEI has one big advantage that it has almost no toxicity whatsoever and also its much cheaper as you can purchase your own PEI-25000 and make the stock solution. So my recommendation would be to go with PEI. All the best.
Dear samarendra ,
Thank you for your answer.
Would you please let me have your exact protocol for PEI transfection?
Best
Niloufar
@SamarendraSingh
Dear Niloufar, sure, give me ur email id and i will forward you the PEI based transfection protocol. Thank you and all the best.
I have gone through your questions, suggestion..i am working on gastric cancer cell, also working on transfection in my primary cell (AGS ) with PEI/Polybrene...same satiation i am facing...as PEI plus Polybrene kill my all cells in very first attempt..in second attempt i try to transfect the cell with PEI with out being use of Polybrene..i got 90% transfection efficiency...later on after 30 hours my cell losing efficiency and start die.
@Samarendra Singh Please share your optimize protocol at [email protected] looking forward.
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