Premium
Engineering of technetium‐99m‐binding artificial receptors for imaging gene expression
Author(s) -
Simonova Maria,
Shtanko Olena,
Sergeyev Nikolai,
Weissleder Ralph,
Bogdanov Alexei
Publication year - 2003
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.448
Subject(s) - transfection , microbiology and biotechnology , membrane protein , receptor , gene delivery , gene expression , molecular imaging , cell surface receptor , biology , gene , chemistry , membrane , biochemistry , in vivo
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. Copyright © 2003 John Wiley & Sons, Ltd.