A faster way to make GFP-based biosensors: two new transposons for creating multicolored libraries of fluorescent fusion proteins.
BACKGROUND
There are now several ways to generate fluorescent fusion proteins by randomly inserting DNA encoding the Green Fluorescent Protein (GFP) into another protein's coding sequence. These approaches can be used to map regions in a protein that are permissive for GFP insertion or to create novel biosensors. While remarkably useful, the current insertional strategies have two major limitations: (1) they only produce one kind, or color, of fluorescent fusion protein and (2) one half of all GFP insertions within the target coding sequence are in the wrong orientation.


RESULTS
We have overcome these limitations by incorporating two different fluorescent proteins coding sequences in a single transposon, either in tandem or antiparallel. Our initial tests targeted two mammalian integral membrane proteins: the voltage sensitive motor, Prestin, and an ER ligand gated Ca2+ channel (IP3R).


CONCLUSIONS
These new designs increase the efficiency of random fusion protein generation in one of two ways: (1) by creating two different fusion proteins from each insertion or (2) by being independent of orientation.
Interdepartmental Neuroscience Program, Yale University, New Haven, Connecticut 06520, USA. douglas.sheridan@yale.edu
Douglas L
DL
Sheridan
Thomas E
TE
Hughes
D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D003116:Color; D004251:DNA Transposable Elements / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000196:embryology / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics*
D004251:DNA Transposable Elements; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins