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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

Tricistronic hNET vector design and ASP+-guided FACS. Three hNET encoding vectors were designed and transduced into SupT1 cells; hNET.l.dCD34 (vector 1), SupT1/FLuc.2A.RQR8.2A.hNET (vector 2), SupT1/hNET.2A.FLuc.2A.RQR8 (vector 3). Transduced (grey lines) and non-transduced (blue lines) cell populations were stained with ASP+ followed by flow cytometry (left column). ASP+-guided FACS was performed by gating on the brightest 5% within each of the three cell populations (right column).
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Fig2: Tricistronic hNET vector design and ASP+-guided FACS. Three hNET encoding vectors were designed and transduced into SupT1 cells; hNET.l.dCD34 (vector 1), SupT1/FLuc.2A.RQR8.2A.hNET (vector 2), SupT1/hNET.2A.FLuc.2A.RQR8 (vector 3). Transduced (grey lines) and non-transduced (blue lines) cell populations were stained with ASP+ followed by flow cytometry (left column). ASP+-guided FACS was performed by gating on the brightest 5% within each of the three cell populations (right column).

Mentions: We constructed a tricistronic vector which allows engineered cells to be tracked in vivo by BLI and SPECT/PET and ex vivo by histochemistry. We co-expressed a bright red-shifted firefly luciferase, along with the hNET and RQR8 - a small marker gene which binds the widely used antibody QBEnd/10 enabling flow cytometry and histochemistry. We used 2A peptide linkage to drive obligate stoichiometric co-expression [26]. Two candidate tricistronic vector constructs were designed and tested; one with 2A peptide linkage at the N-terminus of hNET (FLuc.2A.RQR8.2A.hNET) and one at the C-terminus (hNET.2A.FLuc.2A.RQR8). RQR8 function and transduction efficiency of the two vectors was verified via flow cytometry and QBEnd10 staining. As a reference, SupT1 cells encoding an IRES linked bicistronic vector (SupT1/hNET.I.CD34) were used. Using the optimal staining conditions determined above, ASP+-guided FACS was performed to isolate hNET positive SupT1 cells by gating on the brightest 5% of each population. Post sorting, positive ASP+ staining increased from 59.4% to 99% ± 0.01% for SupT1/hNET.l.dCD34, 46.2% to 93.6% ± 0.34% for SupT1/FLuc.2A.RQR8.2A.hNET, and 52.4% to 93.6% ± 0.66% for SupT1/hNET.2A.FLuc.2A.RQR8 (Figure 2). No difference in growth rates were observed in all cell lines compared to non-transduced SupT1 control cells. hNET function in all cell lines was tested over a period of 17 weeks via ASP+-guided FACS without any significant reduction in uptake observed (Additional file 1: Table S1).Figure 2


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)

Tricistronic hNET vector design and ASP+-guided FACS. Three hNET encoding vectors were designed and transduced into SupT1 cells; hNET.l.dCD34 (vector 1), SupT1/FLuc.2A.RQR8.2A.hNET (vector 2), SupT1/hNET.2A.FLuc.2A.RQR8 (vector 3). Transduced (grey lines) and non-transduced (blue lines) cell populations were stained with ASP+ followed by flow cytometry (left column). ASP+-guided FACS was performed by gating on the brightest 5% within each of the three cell populations (right column).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Tricistronic hNET vector design and ASP+-guided FACS. Three hNET encoding vectors were designed and transduced into SupT1 cells; hNET.l.dCD34 (vector 1), SupT1/FLuc.2A.RQR8.2A.hNET (vector 2), SupT1/hNET.2A.FLuc.2A.RQR8 (vector 3). Transduced (grey lines) and non-transduced (blue lines) cell populations were stained with ASP+ followed by flow cytometry (left column). ASP+-guided FACS was performed by gating on the brightest 5% within each of the three cell populations (right column).
Mentions: We constructed a tricistronic vector which allows engineered cells to be tracked in vivo by BLI and SPECT/PET and ex vivo by histochemistry. We co-expressed a bright red-shifted firefly luciferase, along with the hNET and RQR8 - a small marker gene which binds the widely used antibody QBEnd/10 enabling flow cytometry and histochemistry. We used 2A peptide linkage to drive obligate stoichiometric co-expression [26]. Two candidate tricistronic vector constructs were designed and tested; one with 2A peptide linkage at the N-terminus of hNET (FLuc.2A.RQR8.2A.hNET) and one at the C-terminus (hNET.2A.FLuc.2A.RQR8). RQR8 function and transduction efficiency of the two vectors was verified via flow cytometry and QBEnd10 staining. As a reference, SupT1 cells encoding an IRES linked bicistronic vector (SupT1/hNET.I.CD34) were used. Using the optimal staining conditions determined above, ASP+-guided FACS was performed to isolate hNET positive SupT1 cells by gating on the brightest 5% of each population. Post sorting, positive ASP+ staining increased from 59.4% to 99% ± 0.01% for SupT1/hNET.l.dCD34, 46.2% to 93.6% ± 0.34% for SupT1/FLuc.2A.RQR8.2A.hNET, and 52.4% to 93.6% ± 0.66% for SupT1/hNET.2A.FLuc.2A.RQR8 (Figure 2). No difference in growth rates were observed in all cell lines compared to non-transduced SupT1 control cells. hNET function in all cell lines was tested over a period of 17 weeks via ASP+-guided FACS without any significant reduction in uptake observed (Additional file 1: Table S1).Figure 2

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