Stable transfection of large proteins

Any suggestions for rgts/techniques to ensure stable transfection of large (~14kDa insert) constructs in an adherent cell line.
I'm not having much luck with this - its a 400+ kDA protein!

Yes, this works with 293T with no problems! I'm trying to get it in MDCK now. The protein does have multiple transmembrane domains, as well as pretty large extracellular and internal regions. Have tried everything from Ca-phos to Lipofectamine (and variants from 4 companies!). Did manage to get some very transient transients (picked in screen and by Western first time, but could not repeat), but no stable clones to date.

Have you tried microinjection?

I know that in my former lab, the transfection method of choice was microinjection for MDCK cells.

Another idea is the Nucleofector from Amaxa (www.amaxa.com). It is an upmarket electroporator, preprogrammed with optimized protocols for transfection hundreds of cell lines and primary cultures. I am trialing one at this very moment. I am having stunning success transiently transfecting CHO K1 cells with cDNA (88-90% efficiency). I am not making stables, but it should be a good starting point. The supposed benefit of this device is that it delivers a large proportion of the cDNA to the nucleus and the transfected cell starts expressing within hours, rather than waiting for mitosis and the daughter cells to express. I have seen expression in the parent cell after 3 hours. It may be decetable sooner, but that is the earliest time point I have looked at so far.

Most people I have spoken to have said it is the best thing on the market for strong, efficient, reproducible expression (5 labs, including my formar lab). The only drawback is that the approx $12,000 initial outlay is steep and so are the cuvettes and tranfection solutions, averaging $18 per transfection, which you can only buy as prepackaged kits. But then again what is the cost of something that works vs many attempts at a cheaper solution that does not work as well?

Thanks frasermoss! I came across the AMAXA stuff in a paper, and checked them out - seems workable especially since the "electroporator" is already there in an adjoining dept. However, the company itself seems to have no protocols for MDCK, and the user database has used very small (usually the positive control GFP) constructs. Have you ever tried using nucleoporation on these cells, and if so, any suggestions please, especially with regard to programs used, etc (all users seem to have used solution "T", so that is one thing resolved)!
Also, have you ever tried for large plasmid/stable transfectants at all - any suggestions there will also be welcome.
We do not have either a micromanipuulator or the handling skills, so I guess microinjection is out of the question.
Once again, thanks for your input.

You'll need to set up a free user account with amaxa on their web site then you can access the cell collections wich gives you a list of cell lines, which include MDCK and MDCK purchased from ATCC.

the links are

http://www.amaxa.com/celldetails.html?&tx_mwaxcelldb_pi3%5Bcell%5D=153&no_cache=1

and

http://www.amaxa.com/celldetails.html?&tx_mwaxcelldb_pi3%5Bcell%5D=152&no_cache=1

you will have to be logged into your amaxa account before you can view them though.

I have only trialed the device for a week so far but in addition to various GFP's the largest plasmid I have got in was 10.5 kb, containing a potassium channel in to CHO K1 cells. I got good currents when i did whole cell patch electrophysioogy on the cells the next day. I dont work with MDCK at all

The actual electroporation pulses are all preprogrammed into the nucleporator device for each cell line, so i dont think there is a way to find out size, frequency and duration of the pulses they deliver if you wanted to transfer the technique to a normal electroporator.