It may be feasible to improve FRET output by equalizing donor and acceptor expression, e.g., by placing the components on separate plasmids (we avoided this strategy here because of the need to introduce the target ligand using a second plasmid).
In addition to the irradiated plasmid, we used a second unirradiated control plasmid to correct any effects the transfection process might have on the repair capacity of cells.
Transformed cells were selected on LB agar with ampicillin, and the plasmid was extracted using a plasmid DNA purification kit (NucleoSpin Plasmid, Macherey-Nagel).
Human embryonic kidney (HEK) 293H cells (Life Technologies, Grand Island, NY, USA), preadapted for serum-free suspension culture, were stably transfected with a plasmid containing a dual expression cassette for a FIXFc and Fc chain, a second plasmid expressing Fc alone, and a third plasmid expressing PC5, an enzyme used to ensure complete processing of the FIX propeptide 11, 14.
We used this strain to study GAPDH activity by performing classic 5'fluoroorotic acid (5'FOA) plasmid shuffle assays, in which a second plasmid carrying a HIS3 marker and the gene to be studied were introduced into cells.
First we co-expressed CRE and GFP using a single plasmid containing an internal ribosome entry site (pCAG-CRE-IRES-GFP).
We next co-injected the 3G86.32-mRuby2 encoding plasmid with a second plasmid encoding GFP-CVIM.
A second plasmid was generated which harbors additional upstream sequences.
The PCR products of the N-terminal portion of the domain-swap were used as the forward primers in a second PCR reaction, using a plasmid encoding the other protein as the vector.
First, pcDNA3-transformed DH5α E. coli were grown overnight and the plasmid was purified using a Qiagen Plasmid Midi Kit (Qiagen Inc., Mississauga, ON, Canada).
