Engineering of technetium-99m-binding artificial receptors for imaging gene expression

✍ Scribed by Maria Simonova; Olena Shtanko; Nikolai Sergeyev; Ralph Weissleder; Alexei Bogdanov Jr

• Publisher: John Wiley and Sons
• Year: 2003
• Tongue: English
• Weight: 252 KB
• Volume: 5
• Category: Article
• ISSN: 1099-498X
• DOI: 10.1002/jgm.448

✦ Synopsis

Abstract

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.