Hi Laura,

Did you try using your DNA (complete and empty vector) in other cells? It's possible that there maybe something in your DNA other than the gene that is toxic.

-ED

Hi Laura,

What kit do you use to prep all your plasmid DNA? Bacterial endotoxin might not be removed completely during extraction/purification.

Have you tried other transfection reagent on HeLa cells? I have used XtremeGENE HP DNA transfection reagent and it worked fine on HeLa cells.

I agree with Kai Ling, endotoxins or other high-molecular weight lipopolysaccharides may be the most likely reason for your poor transfection results. Maybe you could change / optimize your DNA extraction procedure (use another kit for example).

Another thing might be to make sure not to grow your bacterial culture for too long. It is better to increase the culture-volume and keep incubation time at the minimum - that will also decrease the endotoxin-levels that are finally present in your plasmid preparation. DNA quality beats quantity!

Also, as mentioned by Emmanuel, try to use some control plasmids from other labs with HeLa or other cells to see if you can improve your transfection-procedure.

Good luck with your experiments,

Christian

Thank you all for your comments. I am currently using the Zyppy miniprep kit and has used the Qiagen one in the past. In my old lab the plasmids prepared with Qiagen were easy to transfect with HEK293T etc and not HeLa. Seeing as the problem is with the addition of any DNA it seems possible that the way the samples are prepared is causing a problem. 

I will be trying to transfect 293T cells next to see how these cells react but it is very frustrating seeing as HeLas are meant to be easy! 

I agree with Dr. Laura Collopy.   HeLa is surely the vital cells than slow-growing 293T cells.  

I have studied and determined the protein amount of invaded microbes by using protein-direct-microsequencing-deciphering method (PDMD method) on two cultured cells, and have been astonished by the unexpected result (see file HepG2 fucoidan).

HepG2   6.6% viral proteins per total cell proteins

Hc      21.9% viral, bacterial, plant's proteins per total cell proteins.

(Interesitingly, fungal proteins are not detected in the case of cultured cells and biopsies of livers, but human serum has fungal proteins (10/12 tested)(data not shown). 

Although, I have not yet determined the amount of DNA and RNA in HeLa cells, HeLa cells may highly be infected by many viral DNA and/or RNA. 

Further, I have not yet determined viral proteins, HeLa may already have a high content of infected viral proteins.

This is why HeLa is most vital cells in the world.

Therefore, the high levels of content of the already existing vira in the cells may destine the success of additive infection or transformation.

In the case of HeLa, another method to introduce the genes by cell fusion may be a possible choice (file Yamamoto Fusion and HeLa Carrot). 

These are the things that you can try-

1. Quickly clone your insert in some other known shuttle vector and try to see if it causes the same pattern of cell death. 

2. As already suggested, purify your DNA carefully to avoid remnants of any bacterial endotoxins. It is quite possible that something in your DNA prep is causing the toxicity. Does your empty vector (prepared by same method) also show similar cell death? You can check that also.

3. Check the timing of cell death. If it is due to your protein's expression, then, death should begin at least after 36 hours after transfection, when your construct would begin to express. But if you see cell death even after 1-2 hours of transfection, then it cannot be due to your gene or any other leaky protein expression. For any protein to exert its toxic effects should exceed a threshold level (number of molecules present in the cell). It is likely that cell will degrade the unwanted proteins quickly, so for substantial accrual a minimum time is required.

4. Try to dilute your DNA (before transfection) in autoclaved and filter sterilized miliq water. Sometimes, salts along with DNA may interfere in transfection.

I will try these suggestions and report back, thank you. I had previously not removed the Lipo/DNA mix from cells because the protocol states there is no need for this but clearly this is not the case. As kindly suggested I will monitor the cells and see when they die but removed the mix after 5 hours. Tias Saha - thank you also for your suggestions, I will try points 1-3. For point 4 I have already diluted my DNA prior to transfection and this did not seem to make a difference. 

Thank you all again, I will see how it goes this time around. 

Your trouble is either coming from your DNA quality, transfection reagent or your cells. I recommend using endotoxin free kit to purify your DNA, use healthy cells (mycoplasma free) with low passage number and finally maybe try another transfection reagent, I can suggest HeLaFect or DreamFect Gold : http://www.ozbiosciences.com/6-transfection

Good Luck