Hi,
I have been trying to transiently express and examine cAMP accumulation of human 5HT4 receptor in HEK293. The signal peptide-flag tagged 5HT4 is expressed on the cell surface, but cAMP accumulation is minimal above background. I have the following questions:
1. Would any one know what is the best cell line to use for transient transfection of 5HT4? I notice that a lot of people use COS-7. Are there particular reason why people use Cos-7 for transient trasnfection of 5HT4 receptors? It seems to take such a large amount of DNA per eleectroporation of COS-7. Plus, my PI doesn't think it's "physiolgically relevant".
2. I tried other cell lines such as CHO-K1 but it requires expensive kit such as AMAXA. Does anyone happen to have an optimized electroporation protocol that doesn't take so much DNA or cost so much for CHOK1 or COS-7?

Thanks in advance.

What kind of electroporator are you using? Manufacturer and cuvette gap size please.

If I have that info I could probably come up with a protocol for you.

Also do you have to use electroporation?

Have you thought about other transfection techniques?

e.g.

Calcium Phosphate
Lipofectamine
Jet PEI
Effectine
Fugene
EcoTransfect

By the way - CHOK1 should be easy to electroporate too and you dont need to go to the expense of using an Amaxa machine and reagents. 100V 50 ms should work just fine with 1 to 4 million CHO-K1 cells in 100 ul buffer (PBS or serum free media). You will have to vary the amount of plasmid you try though to optimize your expression. 5HT4 is a pretty big message compared to something like GFP so experiment.

A good trick is to put the cells in a low calcium media for 30 mins in the incubator immediately after transfection (en RPMI 1640 media) to allow them to recover.

If you want a more Neuronal type cell line why not try Neuroblastoma N2a cells? I dont know if the have any endogenous 5HT4 though. You'd have to check.

PC12's are another alternative

Also try going to

http://www.btxonline.com/applications/register.asp

for a range of electroporation protocols

We use BioRad Gene Pusler XCell and 0.4 cm cuvette. I tried lipofectamine 2000 on CHOK1 and got very low transfection efficiency (5-10%). I haven't tried COS7 yet.

Thanks again.

Peter