Questions on transfection - (Jun/29/2005 )
Hi, all:
I need to construct a GFP fusion vector for my study. Since no one in our lab had past experience on thsi before, I thought I'd better ask for some professional opinions here before proceeding.. 
My protein of interest is a type II transmembrane protein (that is, N terminus in the cytoplasm and C terminus hangs extracellularly). I'm thinking of using Clontech's pEGFP-C1 vector to construct my fusion vector.
1. For the primer design: Is it true that all I need to care about would be having my protein sequence (starting from the 1st codon of it's N-terminal cytoplasmic tail) cloned in frame right after the GFP sequence and nothing else to consider about?
2. For the transfection part: I'm planning to use transfecting reagents for putting the constructed vector into 293T and CHO lines. I've researched on the use of commercial reagents and it seems that most people use either Lipofectamine 2000 or Fugene. Could anyone with experience with these two help me out with their pros/cons (e.g. ease of use, transfection efficiency,...etc.).
3. Finally, how will I be able to do and determine if I get a permenant transfectant or not?
Many thanks in advance for helping a newbie out.. 
generally, amounts of DNA per number of cells is given in the users' manual. I use "standard" lipofectamine and for a 6well plate i use 2µg DNA and 13 µl lipofectamine. Works fine. you may better do transfection tests before...
Permanant transfectant is obtained by antibiotic selection. Cells that have integrated yourvector in their genome and actually stil express the GFP are stable transfectants.
Good luck.
fred
Thanks for your kind reply, Fred. 
I'm thinking of using Clontech's pEGFP-C1 vector to construct my fusion vector.
1. For the primer design: Is it true that all I need to care about would be having my protein sequence (starting from the 1st codon of it's N-terminal cytoplasmic tail) cloned in frame right after the GFP sequence and nothing else to consider about?
answer: yeah that is right...though it depends on how big your protein of interest is. Normally Transmembrane proteins are rather big, so you may have a pretty hard time cloning the whole thing out. if you have the construct already in another vector, you may consider releasing and pasting the construct into the appropriate pEGFP vector. Just chose the one that match your restriction sites and is in frame.
2. For the transfection part: I'm planning to use transfecting reagents for putting the constructed vector into 293T and CHO lines. I've researched on the use of commercial reagents and it seems that most people use either Lipofectamine 2000 or Fugene. Could anyone with experience with these two help me out with their pros/cons (e.g. ease of use, transfection efficiency,...etc.).
answer: for 293T, it is easily done with CaPO4 protocol, you may refer to Current Protocol for Molecular Biology for detailed explanation. CHO cells, I am not sure. Lipo 2000 and Fugene are both pretty easy to use, but major probelm is toxicity to your cells - which is DNA amount dependent. You may want to play around with the transfection conditions.
3. Finally, how will I be able to do and determine if I get a permenant transfectant or not?
answer: for you to select for permenant transfectant - takes about 2wks. you need to use G418 selection (see pEGFP-C1 product details). I would suggest to plate cells sparsely after you transfect them. in your case it would be rather easy to select the clones...though, you may need to work out the appropriate amt of G418 to use.
best of luck...hope this helps...