A Novel Ideal Radionuclide Imaging System for Non-invasively Cell Monitoring built on Baculovirus Backbone by Introducing Sleeping Beauty Transposon

Sleeping Beauty (SB) transposon is an attractive tool in stable transgene integration both in vitro and in vivo; and we introduced SB transposon into recombinant sodium-iodide symporter baculovirus system (Bac-NIS system) to facilitate long-term expression of recombinant sodium-iodide symporter. In our study, two hybrid baculovirus systems (Bac-eGFP-SB-NeoR and Bac-NIS-SB-NeoR) were successfully constructed and used to infect U87 glioma cells. After G418 selection screening, the Bac-eGFP-SB-NeoR-U87 cells remained eGFP positive, at the 18th and 196th day post transfection (96.03 ± 0.21% and 97.43 ± 0.81%), while eGFP positive population declined significantly at 18 days in cells transfected with unmodified baculovirus construct. NIS gene expression by Bac-NIS-SB-NeoR-U87 cells was also maintained for 28 weeks as determined by radioiodine uptake assay, reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot (WB) assay. When transplanted in mice, Bac-NIS-SB-NeoR-U87 cells also expressed NIS gene stably as monitored by SPECT imaging for 43 days until the tumor-bearing mice were sacrificed. Herein, we showed that incorporation of SB in Bac-NIS system (hybrid Bac-NIS-SB-NeoR) can achieve a long-term transgene expression and can improve radionuclide imaging in cell tracking and monitoring in vivo.

a recombinant baculovirus system is crucial for the further improvement of Bac-NIS system in non-invasive molecular imaging in vivo.
Transposon system, also known as mobile genetic elements, is a non-viral, DNA-mediated gene transfer system, which can efficiently insert genetic information into host chromosomes 18,19 . The "sleeping beauty" transposon (SB), originally isolated from Salmonid fish, is a member of Tc1/mariner superfamily of DNA transposon and is reactivated by molecular reconstruction of the elimination of the inactivating mutations in 1997 20 . It consists of two components: a transposon and a transposase which recognizes the inverted repeat containing direct repeated sequence (IR/DR) flanking the transgene in a transposon 19,21,22 . Until now, SB has been proven as an attractive tool of efficient and stable transgene integration both in vitro and in vivo [23][24][25] .
SB100X, one of the hyperactive versions of SB transposases, possesses a 100-fold more active, as well as a higher integration efficiency, into the host genome than the original version 26 . Thus, to cross the barrier of transient gene expression of the Bac-NIS system, we introduced SB100X into the backbone of recombinant baculovirus vector to obtain a novel hybrid system, which combined the advantages of these two systems. To better improve the novel system, the neomycin/geneticin resistance gene (NeoR) was also introduced into the hybrid system (Bac-NIS-SB-NeoR).
We herein evaluated the long-term reporter gene expression mediated by this novel constructed hybrid system and explored the potential application of this hybrid system in cell tracking and monitoring in vivo.

Function determination of NIS protein expressed by Bac-NIS-SB-NeoR-U87 cells.
In dynamic iodide uptake assay (Fig. 3a), the uptake of 125 I in Bac-NIS-SB-NeoR-U87 cells dramatically increased and reached its peak at 45 minutes with no significant decease till 120 minutes. Two independent experiments with triplicates showed consistent results.
In the iodide uptake inhibition assay (Fig. 3b), the radioactivity uptake in Bac-NIS-SB-NeoR-U87 cells was significantly inhibited by various concentrations of NaClO 4 .

Correlation analysis between radioactivity and number of Bac-NIS-SB-NeoR-U87 cells in vitro.
The correlation analysis between the radioactivity of iodide uptake and the number of Bac-NIS-SB-NeoR-U87 cells displayed a high correlation (R 2 = 0.997, p < 0.0001), which provided a potential invasive and quantitative method in evaluating the viable cell number by determining the intracellular radioactivity (Fig. 3c).

Long-term expression of NIS in Bac-NIS-SB-NeoR-U87 cells.
There was no significant change of iodide uptake by Bac-NIS-SB-NeoR-U87 cells for 28 weeks under G418 selection screening (Fig. 4a). The NIS mRNA expression level was maintained through 28 weeks (Fig. 4b) and there was no decrease of NIS protein either as shown by western blotting analysis (Fig. 4c).  (Fig. 6). In addition, the average radioactivity uptake in tumor tissue at 30 minutes in the SPECT images was also presented (Fig. 7a).

Discussion
Since Bac-NIS system cannot integrate target gene into genome to achieve long-term expression 27,28 , its application in non-invasively cell tracking and monitoring in vivo is thus limited. We have introduced SB transposon, which is highly efficient in gene integration 23,29 , to Bac-NIS system to overcome this obstacle.
In order to establish an efficient single plasmid expression system 30,31 , we designed an "all-in one" plasmid, in which a SB100X sequence and the NIS gene sequence is placed outside and between the two IR/DR sites to facilitate the "cut-and-paste" process of transposon. Briefly, the system works in following process: 1. specific binding of SBsase when it recognizes IR/DRs; 2. pairing of a synaptic complex within two ends as well as binding together by SBsase subunits; 3. excising from the donor locus; 4. recognizing the target sequence in genomes and reintegrating at the target locus 19 . The "cut-and-paste" mechanism of SB transposon mediated transgene integration into the genome has been proven by FISH assay 32 .
In current study, according to the results of flow cytometry, eGFP protein expression can last for 196 days with no obvious decline in G418 screened Bac-eGFP-SB-NeoR-U87 cells, which means the feasibility of achieving a long-term transgene expression by this novel hybrid system. Furthermore, a selective resistance gene is also a crucial part of the novel system for maximizing the advantage of application. The high positive proportion of NIS protein evaluated by cell immunofluorescence at the 196 th day as well as no obvious decline of the NIS mRNA and protein level at the 1 st , 7 th , 14 th , and 28 th week in screened Bac-NIS-SB-NeoR-U87 cells, likewise further proved the capability of this novel hybrid system to sustain long-term transgene expression.
Besides, in a dynamic iodide uptake study, radioiodide rapidly increased intracellularly from 0 to 45 minutes, followed by a dynamic equilibrium during 45 to 120 minutes instead of an obvious decline after peaking compared to some studies 5,15 . The mechanism of dynamic uptake equilibrium consists with the equilibrium of intra/ extra cellular ion change in vitro assay, because NIS function relies on the inwardly directed Na+ gradient 1,2 . Combined with the significant inhibition by perchlorate ion, the cloned NIS gene in the new hybrid system has a specific function of accumulating iodide, which is essential for the further application in cell tracking and monitoring in vivo. Furthermore, the iodide uptake assay at various weeks from the 1 st to 28 th week revealed the ability of accumulating iodine did not decline over time, which was consistent with the change of NIS mRNA and protein level.
In vivo SPECT imaging, a pinhole detector instead of parallel-hole detector was chosen due to the higher resolution. The transplanted Bac-NIS-SB-NeoR-U87 tumor cells were clearly imaged at 30 minutes in each imaging (20 th , 28 th , and 43 th day after transplantation), with a T/NT radio (5.30 ± 0.72, tumor/contralateral tissue). The control U87 cells, however, were not detected by SPECT imaging at any time point. Because of the metabolism in mice, the Bac-NIS-SB-NeoR-U87 cells imaging faded gradually with time in each imaging. In the SPECT images, the stomach and bladder were also visible because of the NIS protein existence in the stomach and 131 I clearance from blood by kidneys. However, despite the NIS protein existence in the thyroid, the thyroid imaging was not as clear as the tumor, stomach, or bladder in the SPECT instrument. At 43 days, the Bac-NIS-SB-NeoR-U87 tumor was clearer than the first and second imaging at 30 minutes, due to the tumor growth accompanied by the increased NIS protein. The result of immunohistochemistry also revealed the NIS protein was abundant in the Bac-NIS-SB-NeoR-U87 tumor tissue, which was consistent with the specific tumor imaging in SPECT.
In our study, the novel hybrid Bac-NIS system (Bac-NIS-SB-NeoR) has been proven its capability to sustain long-term transgene expression both in vitro and in vivo. Considering the significant correlation between the radioactivity and cell number in vitro assay, this novel system also has a great potential value for non-invasively quantitatively monitoring transplanted stem cells or immune cells in vivo. Despite the insertion of transgene into the chromosome, the baculovirus-SB hybrid system is safer because of its near-random genomic insertion compared to the retrovirus, lentivirus and AAV-derived vector systems 33 , which revealed an integration bias into transcriptionally active units in the genome [34][35][36][37] .
Nevertheless, there is still a limitation in our novel vector because baculovirus can be rapidly inactivated by serum complement 38 , which may hinder the application of this novel hybrid system in genetic therapy by direct injection in vivo. Therefore, the new hybrid baculovirus-SB system, which can be used for mediating long-term reporter gene expression, may prefer in non-invasively monitoring in molecular imaging.

Conclusion
In the present study, we have constructed a novel hybrid Bac-NIS-SB-NeoR system that can achieve a long-term NIS gene expression both in vitro and vivo, and is ideal for radionuclide reporter gene imaging, especially in cell tracking and monitoring in vivo. Considering the biosafety, low insertional mutagenesis risk, easy manipulation and production, and the long-term expression of transgene, this novel Bac-NIS-SB-NeoR system has great potential in nuclear molecular imaging in the future, especially in cell tracking and monitoring in vivo, as an alternative option for other virus reporter gene imaging systems.

Methods
Plasmids' construction and recombinant baculovirus production. The CMV-eGFP with two sites of XbaI and EcoRI was amplified by polymerase chain reaction (PCR) from pFB-eGFP plasmid 5 and cloned into pT2-BH (Addgene plasmid 26556) backbone to obtain the plasmid of pT2-BH-eGFP. After that, IR/DR-CMV-eGFP with AvrII and NotI sites amplified from pT2-BH-eGFP plasmid, CMV-SB100x with SalI and BstZ17I sites amplified from pCMV(CAT)-SB100x plasmid (Addgene plasmid 34879), pSV40-NeoR with EcoRI, and NheI sites amplified from pcDNA-GLP-1R plasmid (a gift from Shanghai Institute of Materia Medica), were cloned into pFast-Bac HTB vector(previously stored in our laboratory) with corresponding sites to obtain the hybrid plasmid vector of pFast-eGFP-SB-NeoR.
The NIS gene fragment amplified from the pFast-NIS plasmid 5 was cloned into the backbone of pFast-eGFP-SB-NeoR plasmid at the sites of AscI and BspDI to obtain a new plasmid of pFast-NIS-SB-NeoR.
After plasmids were constructed, the production of two recombinant baculovirus carrying eGFP or NIS genes (Bac-eGFP-SB-NeoR and Bac-NIS-SB-NeoR) were prepared as described previously 5 . The end-point dilution 39 and RT-PCR 40 method were used for determining the titers of the two recombinant baculovirus.
G418 screening infected U87 human glioma cells. U87 cells, which were cultured in DMEM medium (ThermoFisher Scientific, Shanghai, China) supplemented with 10% FBS (HyClone, Logan, UT) and 1% penicillin-streptomycin (ThermoFisher Scientific, Shanghai, China) in 37 °C incubator with 5% CO 2 , were used to test the ability of long-term transgene expression mediated by novel constructed hybrid system. The Bac-eGFP-SB-NeoR and Bac-NIS-SB-NeoR virus were added with final multiplicities of infection (MOI) of 0 and 200, respectively. After a four-hour infection, U87 cells were washed twice by PBS and replenished with fresh full medium. Forty-eight hours later, G418 (Sangon, Shanghai, China) at 200 ug/ml was used for screening infected U87 cells to obtain stable cell lines expressing eGFP or NIS gene (Bac-eGFP-SB-NeoR-U87 and Bac-NIS-SB-NeoR-U87).

Dynamic iodide uptake of Bac-NIS -SB-NeoR-U87 cells.
The modified study of dynamic iodide uptake was similar as previously performed 41 except for the difference of a serum-free medium instead of HBSS. The stable Bac-NIS-SB-NeoR-U87 cells were seeded in a 24-well plate with a cell number of 5 × 10 4 . Twenty-four hours later, the seeded cells were replenished with 500 ul serum-fresh DMEM medium containing 3.7 kBq (0.1uCi) Na 125 I (Xinke, Shanghai, China) and 10 uM NaI (Sangon, Shanghai, China) per well. When uptake completed, each well was washed three times with cold PBS and incubated with 500 μ l 1 M NaOH for 15 minutes. The radioiodide uptake was terminated and measured by a γ -counting machine at various time points of 0, 5, 15, 30, 45, 60, 75, 90 and 120 minutes.

Inhibition of iodide uptake by ClO 4
− . Twenty-four hours before the inhibition experiments, the stable Bac-NIS-SB-NeoR-U87 cells were seeded in 24-wells with a density of 5 × 10 4 . Three concentrations (0, 50 and 500 uM) of NaClO 4 , together with 500 ul serum-free DMEM medium containing 3.7 kBq (0.1uCi) Na 125 I, and 10 uM NaI were added per well in each group, respectively. After 45 minutes of incubation in a 37 °C incubator with 5% CO 2 , the radioiodide uptake of each group was terminated and measured by a γ -counting machine.

Correlation analysis between radioactivity uptake and cell numbers in vitro. The stable
Bac-NIS-SB-NeoR-U87 cells were seeded at densities of 1 × 10 4 , 2 × 10 4 , 4 × 10 4 , 8 × 10 4 , and 1.6 × 10 5 in a 24-well plate and incubated at 37 °C with 5% CO 2 for 24 hours. Then 500 ul serum-free DMEM medium containing   3.7 kBq (0.1 uCi) Na 125 I and 10 uM NaI was added per well in each group. After 45 minutes incubation, the radioiodide uptake of each group was terminated and measured as above.
Long-term iodide uptake measurements. At 1,2,3,4,6,8,10,12,16,20,24, and 28 weeks after successfully screening stable Bac-NIS-SB-NeoR-U87 cells, the radioiodide uptake was performed as above each week. 5 × 10 4 cells were seeded in a 24-well plate and incubated at 37°C with 5% CO 2 for 24 hours. Then radioiodide uptake terminated at 45 minutes was performed as above. Male BALB/c nude mice that were 3-4 weeks of age (purchased from the Experimental Animal Centre of Ruijin Hospital, Shanghai, China) were subcutaneously injected in the armpit of the right upper limb with 100 μ l of a suspension of Bac-NIS-SB-NeoR-U87 cells which had been cultured for approximately 28 weeks in vitro, followed by an equal amount of U87 cells in the left.

Reverse-transcription polymerase chain reaction.
A hundred-microliter solution containing 500 μ Ci 131 I was injected into the tail vein of each of 3 tumor-bearing mice at 20 days after the transplantation of tumor cells when the diameter of the tumors reached nearly 0.5 cm. A pinhole detector was used for acquiring an image in SPECT instrument(General Electric Company, WI, USA) at 30, 60, 90, 120, 180, and 240 minutes after the injection of 131 I. Each time point image acquisition was under the same circumstances. The second and third in vivo imaging was performed with the same mice at 28 days and 43 days under the same conditions. The average radioactivity in tumor tissue at 30 minutes corresponding to three SPECT imaging was measured by the GE Xeleris image analysis system. Immunohistochemistry. At 43 days, nude mice were sacrificed by cervical vertebra dislocation, and tumor tissues were fixed in 4% paraformaldehyde for 48 hours, followed by paraffin embedding. Mouse monoclonal anti-NIS antibody(Abcam, ab17795, 1:500) was used at 4 °C overnight in immunohistochemistry, and images of each slice were acquired by a fluorescence microscope. Statistical analysis. All data are presented as the mean ± SD (at least N = 3). Statistical analysis was performed using Student's t-test and One-way Analysis of Variance (ANOVA). A p value of less than 0.05 was considered statistically significant.