I would like to perform functional studies in primary human keratinocytes and I am looking for an expression system that offers high transfection effiency. In principle, an adenovirus-transduced gene expression system might be a good choice, but the main disadvantage is the requirement of a laboratory with biosafety level 2. Yesterday, I stumbled over baculovirus-transduced gene expression, and interestingly, there is no need for BL2. Is there anyone familar with this expression system? Thank you very much in advance
Dear Claus, choosing of expression system will depend on desired protein yield, folding/solubility and modification issues and your experience. You can use baculo system without BSL2 precautions, but still you need to work in sterile conditions in BSL2 cabinet (like with any other cell line). To my mind setting up a baculovirus expression system may be a bit tricky with no prior experience. May be you should try HEK293 cells firstly? They grow very well in both serum-supplemented and serum-free medium (which is important for downstream purification), They are easily transfectable with various techniques: lipofection, calcium precipitation, PEI-mediated tansfection. Ca-precipitation in my hands resulted in close to 100% transfection efficiency, and this method is very cost effective. Moreover you can choose from wide variety of vector with different tags and other features. I have achieved productivity up to 6-8 mg/L in a simple adhesion culture in serum-free medium using pCEP4 vector. This vector contains oriP element from EBV which drives episomal replication in HEK 293 cells. This results in high plasmid copy number per cell and high expression levels. If you need more information - let me know. If you want to start with baculovirus system - I'd recommend to browse Invitrogen's systems like Bac-toBac - they allow fast and easy bacmid generation, besides Invitogen offers cells, medium and other reagents for baculo system. Hope this would be helpful.
Dear Boris,
This is a very good answer, I'd like to thank you for it even though the question was not mine. I found it while looking for an expression system for keratinocytes, and was wondering if you have ever tried baculo in keratinoctes. These cells are notoriously hard to transfect, and if bac works simply with Ca-precipitation, it would very, very helpful!
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