Dear all,

I am looking for a vector for the transient transfection of mammalian cells (i.e. HEK293, optionally CHO) suitable for recombinant protein expression with high yields, and would highly appreciate suggestions! From a client I got an open reading frame cloned into pcDNA 3.1 from Life Technolgies, do you think that this vector would be a reasonable choice?

Thanks in advance for your advice!

With best regards,

Ralf

Hi Ralf,

We have done transient transfections of both suspension HEK293 and CHO-S with regular vectors, such as pcDNA3.1 which you mention and also the pSectag vectors and pcDNA6 from the same company. They do work also in transient transfections even though theydo not contain any extra elements to keep the plasmids in the cells. We do get good expression levels and harvest after 4-7 days.

Best regards,
Malin

Dear Malin,

Thank you so much for your answer! The pcDNA 3.1 (as well as the pSectag and pcDNA6) in my point of view has the disadvantage of several NcoI cleavage sites in the backbone. I would prefer a vector for the transient expression in which it would be possible to clone PCR fragments by NcoI/ BamHI/ EcoRI/ XhoI into the MCS, which would facilitate the sub cloning of open reading frames that were first cloned and tested for expression in e.g. E.  coli and Pichia pastoris.  For Pichia pastoris we modified the pPICZ vector (of Life Technologies)  for intracellular expression by removing a NcoI site in the backbone and exchanging the MCS, and I was wondering to do the same for a vector for transient transfection, if there would be none available yet.

With best regards,

Ralf