Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.
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What is it about Lipofectin/Labfectin that makes them so lethal for insect cells during transfection?
I understand that we are using a lipid and a molecule that can act as a lipid but what do they do to kill the cells so readily? Other sites say it might be the DNA that is causing lethality or that it may just be the density of the cells that is affecting it.
Any thoughts or pathways would be greatly apprieciated because neither my advisor or his technicians can think of a reason why.
Hola, I used lipofectin and lipofectamin to transfec Sf9 cells and I haven´t big problems , I made minor volumes of transfection mix (100-200ul/p35mm)to save because if few viruses were formed giving more time of replication you could get enough population for amplification. But when I knew and use polietilenimine (Jet-Pei) transfections didn´t fail. It ´s easier to use and more cheap. But returning to liposomes I´m writing of memory but supervivence was related with lipofectamine concentration in the mix and I used 4ul of it in the transfection mix. Buena suerte
That's what most people think about it. So in the reaction, the phosphatidylethanolamine (dope) would bind to the plasma membrane and fuse to bring in the DNA, but how does it cause lethality? Does it destablize the membrane while fusing? Another source claims it's the DNA being transfected that is lethal.
Honestly, I have no idea about this I was just guessing, since they are able to interact with the membrane, but I have no expertise in this area and I have never worked with these substances.
Cis or trans? That's what matters.
I don't know.
That, said, like most cells, insect cells are not built for the lab environment. You need to remember that. It has taken someone many years of optimising the conditions which you use to culture them; mess with any of them, and the cell is going to probably die. Remember also that most cells are also designed to curl up their toes by apoptosis if they don't get the right signals that are wanted, or get unexpected signals of a dangerous type. This includes being infected by viruses.
I also don't know the exact mechanism for the cells dying; but i would suggest that puncturing holes in the cell to get the DNA in is going to lead to a loss of membrane polarity, and this alone would make the cells sick; depending on how hardy they are, and how well you can look after them will depend on how likely they are to survive. Of course, you have also blasted them with effectively viral DNA. This itself is toxic, and the cell will be changing its gene expression to destroy the foreign DNA (with its non-eukaryotic methylation pattern) and this may just push the cells over the edge, so to speak.
And most man-made chemicals are toxic so that along with the above should answer your question. If not, try asking the company that sell it as well.
6 posts • Page 1 of 1
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