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

Badar A, Kiru L, Kalber TL, Jathoul A, Straathof K, Årstad E, Lythgoe MF, Pule M - EJNMMI Res (2015)

Bottom Line: In vivo cellular conspicuity was confirmed using sequential bioluminescence imaging (BLI) and SPECT imaging of transduced SupT1 cells injected into the flanks of mice.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 (123)I-meta-iodobenzylguanidine (MIBG) administration.

View Article: PubMed Central - PubMed

Affiliation: Division of Medicine, Centre for Advanced Biomedical Imaging (CABI), University College London, 72 Huntley Street, London, WC1E 6DD UK.

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 (123)I-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.

No MeSH data available.


Related in: MedlinePlus

ASP+staining optimisation of hNET expressing cells. hNET positive (blue line) or non-transduced (red line) SupT1 cells were incubated with increasing [ASP+] from 0 to 1 μM. For each concentration, FACS was performed at 10, 30, 60, 120 and 240 min. Suggested optimal concentration and incubation time is 0.05 μM ASP+ and 30 min.
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Fig1: ASP+staining optimisation of hNET expressing cells. hNET positive (blue line) or non-transduced (red line) SupT1 cells were incubated with increasing [ASP+] from 0 to 1 μM. For each concentration, FACS was performed at 10, 30, 60, 120 and 240 min. Suggested optimal concentration and incubation time is 0.05 μM ASP+ and 30 min.

Mentions: Functional determination of hNET vectors requires costly and complex radiochemistry work. We hypothesised that the fluorescent NET substrate ASP+ could allow determination of hNET function based on flow cytometry. A concentration and time-course titration assay was designed to determine optimised conditions for efficient cell staining and sorting of hNET expressing cells using ASP+. hNET expressing and non-transduced SupT1 cells were compared to quantify non-specific uptake (Figure 1). Adequate discrimination between specific and non-specific ASP+ staining is essential to study efficiency of hNET encoding vector transduction and reporter function and to facilitate isolation of pure hNET positive cell populations. We determined that ASP+ is both specifically and non-specifically taken up by cells (Figure 1). We observed good discrimination of specific and non-specific staining with a two-log shift in fluorescence. Optimal separation between hNET positive and hNET negative cells, using minimum incubation time and ASP+ concentration, was 30 min and 0.05 μM ASP+ (Figure 1).Figure 1


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

Badar A, Kiru L, Kalber TL, Jathoul A, Straathof K, Årstad E, Lythgoe MF, Pule M - EJNMMI Res (2015)

ASP+staining optimisation of hNET expressing cells. hNET positive (blue line) or non-transduced (red line) SupT1 cells were incubated with increasing [ASP+] from 0 to 1 μM. For each concentration, FACS was performed at 10, 30, 60, 120 and 240 min. Suggested optimal concentration and incubation time is 0.05 μM ASP+ and 30 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385325&req=5

Fig1: ASP+staining optimisation of hNET expressing cells. hNET positive (blue line) or non-transduced (red line) SupT1 cells were incubated with increasing [ASP+] from 0 to 1 μM. For each concentration, FACS was performed at 10, 30, 60, 120 and 240 min. Suggested optimal concentration and incubation time is 0.05 μM ASP+ and 30 min.
Mentions: Functional determination of hNET vectors requires costly and complex radiochemistry work. We hypothesised that the fluorescent NET substrate ASP+ could allow determination of hNET function based on flow cytometry. A concentration and time-course titration assay was designed to determine optimised conditions for efficient cell staining and sorting of hNET expressing cells using ASP+. hNET expressing and non-transduced SupT1 cells were compared to quantify non-specific uptake (Figure 1). Adequate discrimination between specific and non-specific ASP+ staining is essential to study efficiency of hNET encoding vector transduction and reporter function and to facilitate isolation of pure hNET positive cell populations. We determined that ASP+ is both specifically and non-specifically taken up by cells (Figure 1). We observed good discrimination of specific and non-specific staining with a two-log shift in fluorescence. Optimal separation between hNET positive and hNET negative cells, using minimum incubation time and ASP+ concentration, was 30 min and 0.05 μM ASP+ (Figure 1).Figure 1

Bottom Line: In vivo cellular conspicuity was confirmed using sequential bioluminescence imaging (BLI) and SPECT imaging of transduced SupT1 cells injected into the flanks of mice.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 (123)I-meta-iodobenzylguanidine (MIBG) administration.

View Article: PubMed Central - PubMed

Affiliation: Division of Medicine, Centre for Advanced Biomedical Imaging (CABI), University College London, 72 Huntley Street, London, WC1E 6DD UK.

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 (123)I-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.

No MeSH data available.


Related in: MedlinePlus