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Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
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primary vs. established cells??
Posted by: mk801 (IP Hidden, New member, 1)
Date: June 8, 2005 04:20PM
Hello, I will be transfecting (ectopically, transiently) my gene of interest to study its localization/interaction with a certain protein. I was originally going to do this in COS7 or HeLa cells, but my colleague does not want to do that. He insists we use primary cells, because they are not as "screwed up" as transformed cell lines.
1) I just need to see whether my protein interacts with this other known protein. I don't care whether it is in a "screwed up" background. I understand his concern, but I don't think its really necessary in this experiment. I would use a regular mammalian expression vector for this. Who is right? Me or him? 2) How difficult is it to transfect primary cells - say, human skin fibroblasts? Would I have to use some other fancy vector to transfect primary cells? I don't want to use Amaxa's apparatus because I don't want to buy all that stuff. I just need a quick and dirty assay about the protein interactions. If it looks promising then we could design some cool fancy experiments for further study. Please help. Soon. Thank you very much in advance.
Re: primary vs. established cells??
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: June 8, 2005 04:36PM
Well, you can argue either way. The most important thing is to prove your point by showing your data....and publish your results in a peer-reviewed journal (with impact factor >5)!!
How about transfecting primary and HeLa/COS cells at the same time with same vector (no need to be retroviral vector, regular CMV vector should do the trick) using the same cell transfection reagent (no need for Amaxa, too expensive). Either one works then you're home free. I know one reagent -Gencarrier-1 can do the job. Our lab (NCI) routinely used gencarrier-1 for transfection of primary human keratinocytes, epithelial and endothelial cells as well as established COS, HeLa, 3T3, CHO, k562 and 293 lines. It works perfectly.
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