Hello Laura,

Do you want to make loss and gain of function experiments in two seperate lines or in only one line? Do you want to use short hairpin vectors for RNAi, vectors to overexpress genes or at least do you want to perform genome editing (CRISPR/Cas9) for your experiments? You must specify question more detailed otherwise your intention is unclear to provide suggests.

This linke might be helpful

https://www.lifescience.roche.com/shop/products/transfection-protocols-for-primary-cells-transfection-primary-cells

Regards

Hello Volker, thank you for your answer. In my experimenti I'd like to make loss and gain of function in the same cell line, and I'd prefer trying with vectors such as plasmis or RNAi as well.

Hi Laura,

I still not 100% sure exactly what you want to do, but seems like you want to reduce expression of a protein in hepG2 cells and restore it with a mutant? Am I correct?

It is possible to target your gene of interest in the 3'UTR with a siRNA and transfect the plasmid with your mutant-protein (No 3'UTR) at the same time. Co-transfections of Plasmids and siRNAs is really easy. You just need to know how much time it takes for your protein of interest to knock down, this will depend on how stable it is. And how much time stays downregulated. For HepG2 I'm pretty sure you can co-transfect DNA/siRNA with Lipofectamine2000.

Like Volker mentioned above, you could Knock Out the gene you want with CRISPR/Cas9, that takes some time but it is a permanent solution. You should look around if someone has a cell line KO of your protein. That would make your life easier.

PS. What is HCC model?

Hope this helps, good luck!

Hi Joaquin, 

My purpose is create a " complete cancer model in vitro" with HepG2 and also other different cell line  (normal cell line better). I guess you know that HepG2 cell are already tumoral, but I'd like to create some mutation, for example upregulation of hTERT, upregulation of MYC, dowregulation of p53 and something like this. the aim is evaluate metabolic alteration associated qith this kind of genetic alteration. I've never done trasfection with cell, so I'm just looking for a easy protocol, with few molecular biology practice, just a vector to create the mutation, the trasfection kit and stop.

Have I been more clear?

Haha Yeah, You have been clearer now.

Well, transfection of HepG2s is not difficult, I would go for Lipofectamine2000 first, in my hands it has been the best transfection reagent.

siRNA transfection is really easy, it just works. 100% of the cells get transfected if your cells are healthy. Try to use as litle lipofectamine as possible so you don't stress the cells too much.

On the other hand plasmid DNA is a hit and miss. Again Lipofectamine2000 would be my first option, cheapest of the lipofectamines and usually get the highest transfection efficiency. If you have access to a flowcytometer the best use of your time would be to try to find the best combination of DNA-lipofectamine for X number of your cells. Use a plasmid that expresses GFP to get the an easy read of the transfection efficiency.

This may sound like a lot of work, but it will save you so many headaches down the road that is totally worth it..

Follow the Lipofectamine2000 protocol it is well explained, really easy to follow.

Hope this helps, Have fun!

Hi Laura, you can transfect HepG2 with DreamFect Gold reagents or with the Magnetofection with give excellent results especially for such cells. I will recommend PolyMag or Magnetofectamine for your cells

http://www.ozbiosciences.com/transfection-dna/49-magnetofectamine-lipofectamine-transfection-kit.html

http://www.ozbiosciences.com/transfection-dna/22-polymag-magnetofection-transfection-reagent.html

Let me know if I can be of any help

Good Luck

Please find cell specific transfection protocols at the following web address

https://www.thermofisher.com/uk/en/home/life-science/cell-culture/transfection/transfection-support/transfection-selection-tool.html