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Open Access 01-12-2015 | Original research

Fluorescence-guided development of a tricistronic vector encoding bimodal optical and nuclear genetic reporters for in vivo cellular imaging

Authors: Adam Badar, Louise Kiru, Tammy L Kalber, Amit Jathoul, Karin Straathof, Erik Årstad, Mark F Lythgoe, Martin Pule

Published in: EJNMMI Research | Issue 1/2015

Abstract

Background

In vivo imaging using genetic reporters is a central supporting tool in the development of cell and gene therapies affording us the ability to selectively track the therapeutic indefinitely. Previous studies have demonstrated the utility of the human norepinephrine transporter (hNET) as a positron emission tomography/single photon emission computed tomography (PET/SPECT) genetic reporter for in vivo cellular imaging. Here, our aim was to extend on this work and construct a tricistronic vector with dual optical (firefly luciferase) and nuclear (hNET) in vivo imaging and ex vivo histochemical capabilities. Guiding this development, we describe how a fluorescent substrate for hNET, 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP⁺), can be used to optimise vector design and serve as an in vitro functional screen.

Methods

Vectors were designed to co-express a bright red-shifted firefly luciferase (FLuc), hNET and a small marker gene RQR8. Genes were co-expressed using 2A peptide linkage, and vectors were transduced into a T cell line, SupT1. Two vectors were constructed with different gene orientations; FLuc.2A.RQR8.2A.hNET and hNET.2A.FLuc.2A.RQR8. hNET function was assessed using ASP⁺-guided flow cytometry. In vivo cellular conspicuity was confirmed using sequential bioluminescence imaging (BLI) and SPECT imaging of transduced SupT1 cells injected into the flanks of mice.

Results

SupT1/FLuc.2A.RQR8.2A.hNET cells resulted in >4-fold higher ASP⁺ uptake compared to SupT1/hNET.2A.FLuc.2A.RQR8, suggesting that 2A orientation effected hNET function. SupT1/FLuc.2A.RQR8.2A.hNET cells were readily visualised with both BLI and SPECT, demonstrating high signal to noise at 24 h post ¹²³I-meta-iodobenzylguanidine (MIBG) administration.

Conclusions

In this study, a pre-clinical tricistronic vector with flow cytometry, BLI, SPECT and histochemical capabilities was constructed, which can be widely applied in cell tracking studies supporting the development of cell therapies. The study further demonstrates that hNET function in engineered cells can be assessed using ASP⁺-guided flow cytometry in place of costly radiosubstrate methodologies. This fluorogenic approach is unique to the hNET PET/SPECT reporter and may prove valuable when screening large numbers of cell lines or vector/mutant constructs.

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Title: Fluorescence-guided development of a tricistronic vector encoding bimodal optical and nuclear genetic reporters for in vivo cellular imaging
Authors: Adam Badar
Louise Kiru
Tammy L Kalber
Amit Jathoul
Karin Straathof
Erik Årstad
Mark F Lythgoe
Martin Pule
Publication date: 01-12-2015
Publisher: Springer Berlin Heidelberg
Published in: EJNMMI Research / Issue 1/2015
Electronic ISSN: 2191-219X
DOI: https://doi.org/10.1186/s13550-015-0097-z

Keynote webinar | Spotlight on sleep in brain health

Springer Medicine

Fluorescence-guided development of a tricistronic vector encoding bimodal optical and nuclear genetic reporters for in vivo cellular imaging

Abstract

Background

Methods

Results

Conclusions

Keynote webinar | Spotlight on sleep in brain health

Springer Medicine

Abstract

Background

Methods

Results

Conclusions

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