Hi Arrhenius. I agree with most of the good advice given here and you have to decide whether you want to go with trying a new technique (electroporation or other transfection product) or to just optimize what you have. In my experience, HeLa cells tend to be quite easy to transfect, so the issue might be (as has been suggested) competition between promoters, and if eGFP is just acting as a reporter, then you can take the advice of Ian and Andreas to reduce the eGFP vector concentration. One thing I'll add is that I usually use very low DNA concentrations (0.01-0.5 ug for 5-9 Kbp vectors) for HeLa cells, since I found good transfection efficiency and some sensitivity of our cells to too much DNA in the transfection mix. You might want to look and see if you are getting a lot of floating dead cells after your transfection (this is of course before any G418 or other selection media). I wanted to know how you knew the low transfection efficiency for your pCl-Neo construct? Are you selecting clones based on the GFP fluorescence or immunofluorescence of your protein of interest? eGFP fluorescence does not guarantee that both vectors have been incorporated for stable expression, so Fatemeh's suggestion using bicistronic vectors is a good way to go but again not 100% guarantee.

If you are selecting clones, one thing to keep in mind is that most HeLa batches tend to be a little resistant to G418. I don't know if this is your case, but this means that you must do your dose-response/killing-curve for your HeLa cells to see what's the optimal G418 concentration and also sometimes you have to wait +2 weeks to have actual selection (killing) of your non-transfected cells.

I normally transfect for 4-8 hours (with OptiMEM or low FBS media) using Lipofectamine2000 (Invitrogen) and then replace to normal DMEM media, and on average get a peak of expression at 48hr with a CMV promoter. For selection, I start my selection at 24hr and continue for 6 weeks (at 3 weeks you already have some stable clones).

fugene6 is an excellent transfection reagent which will allow to decrease the amount of plasmid DNA for transfection (about 1 ug per 150 cm2 dish), while keeping high transfection efficiencies. Another possibility would be to use lentiviral vectors to transduce HeLa cells, achieving transduction efficiencies of close to 100%

jetPRIME is working much better than fugene6 in my hands. You can even ask to the company to send you some to try before deciding to bought it

Thank you very much.

Based on my personal experience, serum has affect in transfection and transduction, I suggest you to use a serum free medium during your transfection. Good luck

Hello

I advice you to use FUGENE 6. In our lab we tried different transfection reagents and to transfect 2 or 3 plasmids, FUGENE was the best one. Use Serum-free media for the transfection mixture and 1:5 (DNA:FUGENE) ratio when transfecting with 2 plasmids.

Good luck

I would suggest to use AMAXA . Works even better than fugene in my hands.

Turbofect works fine, 2.5ug DNA in 0.5ml optimum, mix add 4ul turbofect mix

30min rt then add to the cells plate at 3.10 5 cell/well for 6wells plate

2_3h later remove the medium and add at bio free DMEM

Incubate as needed

Show some toxicity sometime

Thanks!

To:Fatemeh Shamsabadi

I used Opti-Mem during the transfection.

Use electroporation such as neon microporator if available. You can obtain a detailed transfection protocol on the website of life technologies. This method yields 90% GFP positive cells with one vector and should work for cotransfection of two plasmids.

Dear Arrhenius Lin,

I can advice you to use a Bicistronic IRES Vectors which you are able to insert and clone two genes, simultaneously. The IRES sequence will give you the same expression level for both genes, instead of singe vector transfection.

You may also optimization to identify the best concentration for each vector, separately.

Good Luck,

Hello Arrhenius,

Have you tried using Lipofectamine 2000? It works very well (>75% efficiency) in Hela cells with two vectors. What promoters are in your vectors? This could be affecting your expression levels. Lipofectamine 2000 also has a quick optimization protocol on the Life Technologies website. You could easliy optimize with both vectors to find the optimal dose for expression. You should check it out.

http://tools.invitrogen.com/content/sfs/manuals/lipofectamine2000_man.pdf

Hi Arrhenius,

I would firstly look at the number of plasmid molecules that you are transfecting in. If they vary in size then you will have different Mwt's and hence 1micrg of each plasmid will contain different numbers of molecules. Also what promoters are they using? 2 promoters the same will compete for the same transcription factors and other reagents to the potential detriment of one plasmid and thus expression. You could therefore change the ratio of the plasmids used rather than 1:1

good luck

Hi Arrhenius,

When I co-transfect with HeLa cells I tend to mix the two DNA constructs before adding serum-free DMEM and FugeneHD (I use a ratio of 4:1 fugene:ug DNA). However you may want to try optimising for your particular constructs by changing ratios of Fugene:DNA or even different amounts of construct especially since you say one vector has lower expression.

Good Luck

To:Nektaria Andronikou

Both pCI-neo and pEGFP-C1 bear CMV promotors,

Hi Arrhenius. I agree with most of the good advice given here and you have to decide whether you want to go with trying a new technique (electroporation or other transfection product) or to just optimize what you have. In my experience, HeLa cells tend to be quite easy to transfect, so the issue might be (as has been suggested) competition between promoters, and if eGFP is just acting as a reporter, then you can take the advice of Ian and Andreas to reduce the eGFP vector concentration. One thing I'll add is that I usually use very low DNA concentrations (0.01-0.5 ug for 5-9 Kbp vectors) for HeLa cells, since I found good transfection efficiency and some sensitivity of our cells to too much DNA in the transfection mix. You might want to look and see if you are getting a lot of floating dead cells after your transfection (this is of course before any G418 or other selection media). I wanted to know how you knew the low transfection efficiency for your pCl-Neo construct? Are you selecting clones based on the GFP fluorescence or immunofluorescence of your protein of interest? eGFP fluorescence does not guarantee that both vectors have been incorporated for stable expression, so Fatemeh's suggestion using bicistronic vectors is a good way to go but again not 100% guarantee.

If you are selecting clones, one thing to keep in mind is that most HeLa batches tend to be a little resistant to G418. I don't know if this is your case, but this means that you must do your dose-response/killing-curve for your HeLa cells to see what's the optimal G418 concentration and also sometimes you have to wait +2 weeks to have actual selection (killing) of your non-transfected cells.

I normally transfect for 4-8 hours (with OptiMEM or low FBS media) using Lipofectamine2000 (Invitrogen) and then replace to normal DMEM media, and on average get a peak of expression at 48hr with a CMV promoter. For selection, I start my selection at 24hr and continue for 6 weeks (at 3 weeks you already have some stable clones).

I forgot to add that if you are not doing transient transfections, then perhaps you can switch to a different secondary vector for your protein of interest, one that has a different selection gene, like hygromycin?

Thanks!

the efficiency with which different plasmids are expressed after co-transfection has little to do with different transfection efficiencies of the separate plasmid DNA molecules because the lipoplexes formed with cationic liposomes (e.g. Lipofectamine 2000) and DNA molecules (and remember that there are hundreds or thousands of DNA molecules incorporated into each lipoplex particle) do not discriminate between different DNA sequences of plasmids over a relatively large size window. In other words there should be no differential incoproration of different constructs into cells. It will be the expression of the mRNA and proteins that are different, as pointed out already.

One can even perform subsequent transfections of cells to further increase transfection efficiency. e.g. perform simultaneous plating/transfection on day0 (immediate transfection of newly trypsinised - or even better Accutase detached - cells can be more efficient and saves one day) then change media and perform another transfection of the now adherent cells and then change media 4-5 hours after the 2nd transfection and leave overnight. We now do this routinely and it works well.

ScreenFect shows improved efficiency compared to Lipofactamine 2000 or LTX for many cell lines. It also has the advantage that, generally, less plasmid DNA is required.

You can request a free sample at www.Incella.com.

Hi Arrhenius,

we have looked into this. The results can be found on

http://www.synvolux.com/co-transfection-reagent

Free samples are on request

Good Luck

First of all I would recommend using equal molar ratios of plasmid if you are trying to achieve comparatively equal effects. Using lipofection reagents specific to HeLa cells (https://altogen.com/product/hela-transfection-reagent-cervix-adenocarcinoma-cells/) will also help you improve the transfection efficiency. Testing a range of plasmid concentrations in a pre-run can help you determine the optimal conditions necessary if you want to generate a stable population of cells.