Detection of dual-gene expression in arteries using an optical imaging method.
We evaluate the in vivo use of an optical imaging method to detect the vascular expression of green fluorescent protein (GFP) or red fluorescent protein (RFP), and to detect the simultaneous expression of GFP and RFP after transduction into arteries by a dual-promoter lentiviral vector driving their concurrent expression. This method involves using a charge-coupled device camera to detect fluorescence, a fiber optic probe to transmit light, and optical filters to distinguish each marker. In animal models, these vectors are locally delivered to target arteries, whereas the gene for a nonfluorescent cell-surface protein is transduced into contralateral arteries as the sham control. The images show distinct areas of bright fluorescence from GFP and RFP along the target arteries on excitation; no exogenous fluorescence is observed in the controls. Measured signal intensities from arteries transduced with the single- and dual-promoter vectors exceed the autofluorescence signal from the controls. Transgene expression of GFP and RFP in vivo is confirmed with confocal microscopy. We demonstrate the use of an optical imaging method to concurrently detect two distinct fluorescent proteins, potentially permitting the expression of multiple transgenes and their localization in the vasculature to be monitored.
Chen
Hunter H
HH
Johns Hopkins University, Department of Biomedical Engineering, Baltimore, Maryland 21205, USA.
Zhan
Xiangcan
X
Kumar
Ananda
A
Du
Xiangying
X
Hammond
Holly
H
Cheng
Linzhao
L
Yang
Xiaoming
X
D000818:Animals; D005263:Femoral Artery / Q000166:cytology* / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D055095:Optics and Photonics / Q000295:instrumentation; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine; D014018:Tissue Distribution; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein