I agree that optimization of cell/transfection reagent is essential, but there is something else to consider. Even though you are referring to the pcDNA3.1/HisB plasmid as "empty," it is far from that. This vector is going to constitutionally express a neomycin resistance gene as well as a short sequence that contains the 6X His tag, Xpress epitope, and EK site regardless if you have your gene of interest cloned in or not. In other words, even the "empty" vector is going to be over-expressing a LOT of protein in the cell as these are CMV and SV40 promoter driven constructs in the vector. This alone could be inducing a protein unfolded response (ER stress) in your cells, or even consuming a majority of histidine (an essential amino acid that is limiting in tissue culture).

My recommendation, in addition to optimizing your ratios, is to attempt to transfect in a different "empty" vector. Use one that does not have a resistance gene, and one that does not have a Tag region to see if this improves survival.

Good luck,

~Adam

I dont think the vector could kill Head and Neck carcinoma, These cell line are very resistant and aggressive. Please revise your Transfection reagent. These reagent are toxic for cells

I believe you should optimize the cell number for the experiment. Please correct me if I am wrong but I think the confluence recommended for transfection is due to cell death after transfection.This is also important for the reproducibility of your experiment.Try to transfect cells between 60% and 70% of cell confluence, which means that they are in a well proliferating state. To re-plate cells 24h before transfection is also recommended.

I also would prefer to plate new cells to avoid contamination.

If you are using electroporation, try short pulses (microsecond to ms) with reduced voltages. If you are using Transfection reagents like Lipofectamine or FuGene, make sure the concentration ratio between reagent:plasmid is the standard (3:1).

We had a similar experience using empty vectors. We have observed that transfection of empty vector using lipid induces cell death and secretion of some soluble proteins including cytokines. We ended up that the unexpected fact is caused as an innate immune response. We and other have reported that transfection using any DNA induces the immune response in several cell types including cancer cell lines and primary cells (even the cells are not immune cells). The transfected DNA is recognized via cytosolic DNA sensors ( cytosolic DNA binding proteins) and induces multiple gene activation along with production of proteins [J. Immunol. 186: 4541-4545 (2011)]. I do not know your cell well, but one of possibility in your case may be similar with our experience. A change of transfection method may reduce the immune activation.

Thanks for your suggestions. The transfection reagent (Lipofectamine 2000) does not appear to be the problem. I did a survival curve to determine how much the cells can tolerate and our transfection controls (lipofectamine only) do not die. It is only the cells that receive the vector. We don't have many choices in terms of transfection method but we could try a different lipid reagent. I'll make sure the reagent:dna ratios are ok and try some different cell concentrations.

I agree that optimization of cell/transfection reagent is essential, but there is something else to consider. Even though you are referring to the pcDNA3.1/HisB plasmid as "empty," it is far from that. This vector is going to constitutionally express a neomycin resistance gene as well as a short sequence that contains the 6X His tag, Xpress epitope, and EK site regardless if you have your gene of interest cloned in or not. In other words, even the "empty" vector is going to be over-expressing a LOT of protein in the cell as these are CMV and SV40 promoter driven constructs in the vector. This alone could be inducing a protein unfolded response (ER stress) in your cells, or even consuming a majority of histidine (an essential amino acid that is limiting in tissue culture).

My recommendation, in addition to optimizing your ratios, is to attempt to transfect in a different "empty" vector. Use one that does not have a resistance gene, and one that does not have a Tag region to see if this improves survival.

Good luck,

~Adam

Thanks Adam! That is a really good point. My husband had suggested something along those lines as well. We will definitely give this a shot.

I routinely purify my maxiprep DNAs through double banding on cesium chloride density gradient centrifugation. This removes all kind of contaminating nucleases and eliminates toxicity problem.

Also, sometimes using too much of empty vector may cause killing of transfected control cells.

Try different transfection reagents and different total amounts of DNA. That will likely solve your problem.

Both Adam and Andrei brought up important points. I have tried reverse transfection myself a few times like what Andrei has said using Lipofectamine LTX and it worked well. Also, I am now using less Lipofectamine than I used to use with similar efficiacy. Sometimes too much transfection agent used, hoping to get better transfection rate, ends up with a lot of dead cells. I have used pcDNA3.1 and PiRES for different experiments and had no much luck with pcDNA myself. Try another plasmid if you can. Good luck.

hi

1- please prepare clean and sterile DNA with EndoFree Plasmid Maxi Kit.

2-you can used electroporation method for transfection .

3-if you have pCDNA3.1 vector in lab , its good control for your works

maybe the gene is not an effective tumor suppressor. Additionally, contamination may always be an issue.

endotoxin could be one reason but the selective drug you probably use for selecting transfectants could be another. Did you try the drug alone on the non transfected cells ?

I suppose you are transfecting with plasmids obtained with a endotoxin-free kit, isn't it?

very interesting.......

how the cells are dead? -were they detached from the bottom of the plates? may have been induced the production of killer factors? to test this hypothesis I simply suggest to collect the culture liquid in which where the killed the cells and to add it to a culture of your cells and see what happens.

Many transfection reagents are harmful to cells. You can try different transfection reagents. There several companies claim their reagents are non-toxic to cells.

Or when you do transfection, you'd better use cell culture medium that doesn't contain antibiotics. Also without serum, might be good for your cells.

Simple answer is that DNA, especially mixed with transfection agents (lipid-based, like Lipofectamine or Fugene, or others) is toxic for cells. That is why there is always a limit of how much DNA you can use. Simple rule of thumb is that you want to be below ~80% of official maximum to keep you cells happy. Moreover, some cells are more sensitive to toxicity of DNA/transfection agent than others.

Whenever we were interested in transfecting cells, we first performed a 'kill curve' using the antiobiotic/drug in combination with the "empty" plasmid to determine the optimum amount. As previously stated, large amounts of DNA are toxic; not just the total ngs but the total base count (eg. Less DNA if plasmid is large, etc.). We usually saw increased killing in cells transfected with the so-called empty plasmid over no-plasmid control cells, regardless of whether the prep was endotoxin-free.

Obviously, changing the ratio of transfection reagent:DNA as well as trying other transfection reagents are good suggestions and most companies will send you a free sample of their reagent to try. Cheers & good luck!

Hi Catherine,

I agree with many of the previous answers but also want to add that different methods requested different amount of your transfected DNA (0,5-20 μg). We hade the same problem many years ago and when we titrated the DNA and found just a few μg was enough (1-2 μg). Try that.

Good Luck

For your next transfection, I would do:

- transfection control without DNA. Just to be sure that it's not your transfection product/protocol that is toxic to our cells.

- transfection with another 'empty' plasmid, to be sure that it's not your empty plasmid that is toxic.

And yes, use an endotoxin free kit to prepare your DNA plasmid.

I am doubtful that there could be endotoxin contamination. I strongly feel that you must examine the vectors and am quite sure, that you will soon find the vectors are not TRULY EMPTY. Try it and it will surprise you.

I think its is your transfection method/protocol.

Do a dose response or start using the lowest amount mentioned by the manufacturers and then keep increasing it until the cells can survive and express protein of your interest. I would also recommend using lower quantities of DNA. Hope this helps. Good luck!

We always include a control with transfection agent alone so that is not the problem. We used an endotoxin-free kit for purification. We are working on trying lower DNA concentrations. Hopefully that helps! Thanks everyone for the suggestions. =)

Dear Catherine,

I guess you are precipitating DNA prior to using for transfection experiments. In case you are using Ammoniumacetate replace it with Sodium acetate because even trace amonuts in the redissolved DNA may be cytotoxic.

Good luck

Alois Palmetshofer

I will check the transfection reagent first

Hi Adam, great suggetion. I would reccomend to check the qality of yr DNA prep and titrate the concentration. Seed more cells.Goo

d Luck

As Ophelia Granio said, endotoxin should be the main reason. Try a endotoxin-free kit for plasmid preparation. Any other contaminants from the plasmi prep may also be toxic to the cells.

I've had issues with pcDNA3.1 previously. We changed vectors and had no issues.

There maybe a micro-competition between the CMV promoter in the vector and the natural promoter of essential gene in the cell line.

Did you try to transfect your plasmid in another cell line (control)? What's your transfection reagent?

Did you try to use different transfect reagents? such as TransIT®-LT1 or TransIT®-2020. And you can use much less Lp2000 than the protocol says.

It could be placebo effect..........just for fun. Till I get brilliant answer!

Did you compared your cell viability with the transfection reagent control. Most of the transfection reagents are cytototoxic so always include reagent control. Also ensure that the plasmid preparation is free from endotoxins. The expression of plasmid encoded resistance marker gene in the empty plasmid might affect the cell viability. Also the time at which you are comparing the results might give diff results. At early time points, its mainly the toxicity of the transfection reagent and the activation of general cellular defense mechansim against the plasmid might reduce your cell viability. But at later time points the encoded proteins show their effect.

Hello all, I would like also to ask about another thing, I used pCMV6-entry plasmid to overexpress my gene (which I want its role in inhibiting influenza virus replication) in alveolar epithelial cells A549 cells, I used empty vector as a control, but I found that even the empty vector could reduce the influenza virus replication as much as my complete construct do, my question is> does anybody work with pCMV6-entry plasmid and got some induction of cytokines that could also attenuate the virus replication?

Hi

Some cells react very aggressively to transfection of plasmids or siRNA by ramping up their IFN response. Check your "empty vector" cells for MxA and/or OAS expression.

Good luck

G

Answer may be more simple reflecting artifacts - impurities in the vector and or excessive dose of the vector dsirupting the target cell.

Adam's answer explains the main cause of your cells dying. There could also be additional issues affecting your cells, including the transfection reagent. We like to have controls of empty vectors, and separate controls of the just the transfection reagent to see if the concentration is too high. Head and neck cancer cells are generally difficult to kill (that's why there's high transfection efficiency with lipofection: https://altogen.com/product/neuro-2a-transfection-reagent-neuroblastoma-cells-ccl131/) but you could still have used a concentration too high, and that could lead to cell death.