Trouble transfecting particular construct - (Oct/20/2006 )

I have just started a new post, picking up from someone else's work (eek!).

One of the things I would like to do is establish a stable cell line expressing my protein of interest fused to GFP. I am using a SH-SY5y clone. They have successfully transfected this cell line with GFP alone and also with another GFP fusion protein using both lipofectamine and some machine called a nucleofector. I am not familiar with it, but I think it is a type of electroporator. So the issue seems to be with expression of the actual construct. It is very, very, very poorly expressed and in those few cells with fluoresence the localisation is not at all like that of the endogenous protein. I've had a few thoughts.

1) They are using GFP Fusion TOPO TA as an expression vector which I have no experience with. I've always used clontech vectors, but presume that since this vector worked no its own and with the other protein it is OK. They tell me it is in frame etc. I could try a clontech vector but that may be pointless (and expensive).

2) They are transfecting with 2ug of DNA. I've considered trying less if that is the problem?

3) Make an inducible cell line, but the cells don't die - just don't express so I don't know if that will help and could be time consuming.

4) Try making a construct with another tag - like HA. I think they've tried MYC and possibly FLAG tagged. MYC gave a different localisation to both endogenous and the GFP construct.

Any ideas? Boss would like me to do an IP. I'm trying with endogenous protein first, but my predecessor didn't have much luck with that so ideally I'd like a cell line.

Thank you,

Rachelle

I understand that u want to localise a protein in cell culture.

Having a HA tagged protien might b better to localise than a GFP fusion protien.

Using 2 or 3 ug or less of DNA for transfection doesnt matter when localisation is the issue. As long as u get the DNA into the cells thats what matters.

Onething to remember is that with transient transfection, the amount of DNA could b high and might alter the localisation of any protien due ot over expression. Therefore, i would advise stable cells for this purpose.

If u do want to make stables, better to start with a easily transfectable cell line like 293's before trying in SHSY5Y, which is a bit more difficult. if it works in ur lab, then its all right, just a suggestion.

if u have doubts abt the plasmid, get it sequenced or look at the sequeced data. better have it confiormed before proceeding.