Hey, i want to measure cAMP in insect (cockroach/hawkmoth) cell cultures, either in primary cell cultures of neurons (preferred) or in stable embryonic cell cultures. I have plasmids for a cAMP Sensor for mammalian expression (pcDNA3.0-Epac). I have only worked with PEI transfection of HEK cells and electroporation of E.coli before. If i understand the baculovirus system correctly, it could be used for the expression of the protein but the cell is then filled with virus and unsuitable for live cell imaging because its physiologically not healthy ? Lentivirus was recommended to me but i cant find the differences to what i read about the baculovirus system. So far our best idea is purification from HEK cells and microinjection of the cells via patch clamp method which would be really time consuming, if any of you could help with ideas for transfection/transduction of the plasmid i would be very thankful!
P.S. would be great if the method could be used in a S1 lab.
Unlike baculovirus lentivirus will integrate your gene into the genome. You can play with the MOI in order to achieve a low number (or even single) of integrations per cell.
You also need to check that your plasmid is compatible with expression in insect cells.
Some lentiviral systems are VLPs where a helper plasmid that is not packaged is used for replication and packaging of a plasmid with your gene of interest. This plasmid also does not integrate into the genome.
You can use both PEI and electroporation for insect cells. Calcium phosphate nanoparticles are also an option.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts