Novel NMR approach to assessing gene transfection: 4-fluoro-2-nitrophenyl-beta-D-galactopyranoside as a prototype reporter molecule for beta-galactosidase.
Gene therapy holds great promise for the treatment of diverse diseases. However, widespread implementation is hindered by difficulties in assessing the success of transfection in terms of spatial extent, gene expression, and longevity of expression. The development of noninvasive reporter techniques based on appropriate molecules and imaging modalities may help to assay gene expression. 4-Fluoro-2-nitrophenyl-beta-D-galactopyranoside (PFONPG) is a novel prototype NMR-sensitive molecule, which is highly responsive to the action of beta-galactosidase (beta-gal), the product of the lacZ gene. The molecule is stable in solution and with respect to wild-type cells, but the enzyme causes very rapid liberation of the aglycone, accompanied by color formation and a 19F NMR chemical shift of 5-10 ppm, depending on pH. Since the product is pH-sensitive, this opens the possibility for direct pH determinations at the site of enzyme activity. Molecular and 19F NMR characteristics of PFONPG in solution, blood, and prostate tumor cells are presented. This prototype molecule facilitates a novel approach for assaying gene activity in vivo.
Department of Radiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9058, USA.
Weina
W
Cui
Pieter
P
Otten
Yingming
Y
Li
Kenneth S
KS
Koeneman
Jianxin
J
Yu
Ralph P
RP
Mason
D000256:Adenoviridae / Q000235:genetics; D001236:Aspergillus oryzae / Q000201:enzymology; D003124:Colorimetry; D003587:Cytomegalovirus / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D005462:Fluorine Radioisotopes; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D058574:Integrin-Binding Sialoprotein; D007763:Lac Operon / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008297:Male; D009598:Nitrophenylgalactosides / Q000097:blood; D011401:Promoter Regions, Genetic / Q000235:genetics; D011471:Prostatic Neoplasms / Q000235:genetics; D019275:Radiopharmaceuticals; D012795:Sialoglycoproteins / Q000235:genetics; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics*
D005462:Fluorine Radioisotopes; C546871:IBSP protein, human; D058574:Integrin-Binding Sialoprotein; D019275:Radiopharmaceuticals; D012795:Sialoglycoproteins; D009598:Nitrophenylgalactosides; D001616:beta-Galactosidase