Engineering of technetium-99m-binding artificial receptors for imaging gene expression.
BACKGROUND
Optimization of gene therapy protocols requires accurate and non-invasive quantification of vector delivery and gene expression. To facilitate non-invasive imaging of gene expression, we have genetically engineered 'artificial receptors', i.e. membrane proteins that bind (99m)Tc-oxotechnetate ((99m)TcOT) via transchelation from a complex with glucoheptonate. The latter is a component of a widely used clinical imaging kit.


METHODS
The engineered marker proteins were designed as type I and II membrane proteins and consisted of (1) an (99m)TcOT-binding domain, metallothionein (MT), and (2) a membrane-anchoring domain. Engineered constructs were used for transfection of COS-1 and 293 cells; the expression of mRNA was verified by RT-PCR.


RESULTS
Immunofluorescent analysis, cell fractionation and immunoblotting revealed expression of marker proteins on plasma membrane. Transfection of cells resulted in strong positive staining of plasma membrane with anti-His-tag antibodies. Scintigraphic imaging in vitro confirmed the ability of transfected cells to bind (99m)TcOT. The fraction of bound radioactivity reached a peak (3.53%) when 0.93 MBq (99m)TcOT was added to transfected COS-1 cells. The experiment-to-control signal ratio was equal to 32 at the same added dose.


CONCLUSIONS
(1) Both types of engineered 'artificial receptors' were expressed on the surface of eukaryotic cells; (2) marker proteins were functional in binding (99m)TcOT; and (3) type II membrane proteins were more efficient in binding (99m)TcOT than type I proteins. We anticipate that the developed approach could be useful for 'tagging' transfected cells with (99m)TcOT enabling imaging of tracking in vivo transduced cells or cell therapies.
Simonova
Maria
M
Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital, Bldg. 149, 13th St., Charlestown, MA 02129, USA.
Shtanko
Olena
O
Sergeyev
Nikolai
N
Weissleder
Ralph
R
Bogdanov
Alexei
A
D002462:Cell Membrane / Q000000981:diagnostic imaging* / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling / Q000379:methods*; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D011877:Radionuclide Imaging; D011956:Receptors, Cell Surface; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013667:Technetium / Q000493:pharmacokinetics*; D014162:Transfection
D011956:Receptors, Cell Surface; D013667:Technetium