How dense were your cells? Density / proliferation status strongly influences transfection efficiency. I have no experience with XtreamGene, but it's said to have very little toxicity... Are you sure it's not your protein that kills the cells?

In our hands the most efficient transfection reagent that we have tested for NIH3T3 is so far JetPEI.

If you want stable transfected clones, the best way to do this is with a retrovirus or lentivirus,... I usually get more than 90% stably transduced cells using retroviruses. If you use a transient transfection method as you did, keep in mind, that only around 0.01% of your GFP positive cells have the plasmid DNA stably integrated in the geneome. Usually you need a good transfection efficiency to get stable transfected clones.

Maybe you should try electroporation. In our lab we get up to 80% transfection efficiency even with hard to transfect cells such as primary endothelial cells and fibroblasts.

Hi Olivier,

answering your question about the density of NIH3T3, they wer at 70-80% of confluence. It is likely that the overexpression of my nuclear protein has a toxic effect, because GFP positive cells died 24 h after transfection. However, previous that observation my problem is that the transfection efficiency is as low as 10%.

Could you tell me about your protocol with JetPEI?

Thanks in advance

Dear Elisabeth,

For my experiments is no necessary to integrate my plasmidic DNA in the genome. However, if using retroviruses it is possible to increase the transfection efficiency, and also obtain estable transfected cell lines, that will be great for me.

I have no experience tranfecting with retrovirus or lentivirus. Could you tell me some advice and procedures for NIH3T3?

I have had reasonable luck in trasfecting 3T3 with plasmids using Lipofectamine 2000, and better luck with Mirus TransIt 3T3 reagent. The latter is specific for 3T3 cells, and worked very well the last time I used it.

Electroporation is good idea... Ratios 3:1 and 6:1 were 6 ul Xtream and 2 ug pDNA or 12 ul Xtream and 2 ug pDNA??? According to hp use the max ratio 1:4 (pDNA:Xtream HP). Did you use serum free medium?

Hi José,

For JetPEI, we followed the protocol from the manufacturer.

If your protein is indeed toxic, then I'm afraid you'll never manage to get stable clones, unless you switch to an inducible system (Tet or rheoswitch). Or, if the toxicity is due to a very high expression level (too many copies and strong CMV promoter of pIRESneo), then you might want to try a weaker promoter or indeed, as Elisabeth suggested, change to virus transduction to try and control the copy number by playing with the MOI.

Normally, 3T3 are among the easiest cells to transfect! As suggested, you can check the density of your cell population the day of the transfection, and check the serum in your medium (whether the Xtreme requires a serum-free medium). The antibiotics in the medium can also be harmful for the transfection efficiency. Normally, those fibroblasts are not fragile, but you might consider using a less toxic transfection reagent such as FugENE (from Roche).

Dear Jose, you're right, for transiently transfected cells it's not necessary that the Plasmid DNA is integrated in the genome. But since you wrote about clones I assumed that you wanted to generate stably transfected cell lines.

If you want to use retro- or lentiviruses you first need an appropriate packaging cell line: e.g. Phoenix-gp (for retroviruses) or 293T (suitable for both). Then you transfect these cells with Plasmids: exprssion plasmid, plasmid for capsid (gag/pol) and envelope expression plasmid (VSVg) using calcium phospate. The supernatant containing the virus is the harvested and you icubate your NIH3T3 with the supernatant for 6 to 8 hours, change the medium, wait 2 to 3 days and then do your experiments. I can send you a detailed protocol if you want.

Dear all,

I''m performing some transient transfection experiments in NIH3T3 using the Lipofectamine 2000 and Lipofectamine siRNAMAX (as Dr. Bradbury suggest me in your answer) because I have these reagents in my lab. If the transfection does not work, then I will buy the JetPEI as Olivier told me. However, I will try to generate stably transfected cell lines in a near future. Elisabeth, could you be so kind for sending me your protocol?

I will inform all contributors about my results.

Thank you again

How do you measure your transfection efficiency? Just based on the fluorescence?

Hi everybody,

thank you very much for your answers.

Anne-Sophie, I have evaluated the transfection efficency using fluorescence. Do you think is better to use a qPCR?

Olivia, thanks for your offer. I will tell to my PhD students to ask you about the advantages to use viromer Red and Yellow to improve the eficiency of our transfections.

Best wishes,

José Luis

Yes, maybe you coulb get a better idea with a qPCR to measure GFP, and do a time course to see when this fluoresceence is higher. Based on experience, the highest expression is usually 2 days after the transfection, but it may vary depending on the cell type. The way you actually do your transfection complexes is also very important.