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

Abstract

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 18^th and 196^th 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.

Figure 1. The schematic of constructed plasmids.

The eGFP and NIS gene both driven by a CMV promoter were inserted into two IR/DR sites whereas SB100X driven by a CMV promoter was outside the two IR/DR sites in a single plasmid. CMV promoter = cytomegalovirus promoter, IR/DR = inverted repeat/direct repeated sequence, SB100X = Sleeping Beauty 100X.

Figure 2. The long-term eGFP and NIS gene expression mediated by the novel hybrid system.

The positive proportion of transgene expression at the 196^th day after G418 screening in Bac-eGFP-SB-NeoR-U87 (a) and Bac-NIS-SB-NeoR-U87 (b) cells was observed by fluorescence microscope. No significant decline (one-way ANOVA, p = 0.41) of eGFP+% from the 18^th day to the 196^th day in G418 screened Bac-eGFP-SB-NeoR-U87 cells was determined by flow cytometry (c), whereas a significant decline (one-way ANOVA, p < 0.0001) in other baculovirus infected U87 cells (c). eGFP+% = the percentage of eGFP positive cells, Data = means ± SD (at least N = 3).

Figure 3. The specific function assay of NIS protein in screened Bac-NIS-SB-NeoR-U87 cells.

The dynamic iodine uptake curve in screened Bac-NIS-SB-NeoR-U87 cells (a) and significant (two tailed Student’s t-test, p < 0.0001) inhibition effects by NaClO₄ at various concentrations (b). A significant (p < 0.0001, R² = 0.997) correlation between radioactivity and cell number of screened Bac-NIS-SB-NeoR-U87 cells (c). “***” = p < 0.0001, NS = no statistically significant difference. Data = means ± SD (at least N = 3).

Figure 4. The long-term NIS expression assay.

No significant (one-way ANOVA, p = 0.81) decline of NIS gene expression in screened Bac-NIS-SB-NeoR-U87 cells was determined by radioiodide uptake (a), mRNA level (b) and protein level (c) in various weeks. Lane 1, 2, 3, and 4 means 1^st, 7^th, 14^th, and 28^th week. Data = means ± SD (at least N = 3).

Figure 5. The dynamic imaging by SPECT instrument.

The imaging variation over time in the first SPECT imaging (20^th day after transplantation with screened Bac-NIS-SB-NeoR-U87 cells). The green and red circle respectively represent the Bac-NIS-SB-NeoR-U87 cells and U87 cells. Blue, gray and yellow arrows respectively represent thyroid, stomach and bladder.

Figure 6. Three imaging at different days after transplantation with screened Bac-NIS-SB-NeoR-U87 cells by SPECT.

The successfully detecting of Bac-NIS-SB-NeoR-U87 tumor cells at 30 minutes in each SPECT imaging in vivo. The green and red circle respectively represent the Bac-NIS-SB-NeoR-U87 cells and U87 cells. Blue, gray and yellow arrows respectively represent thyroid, stomach and bladder.

Figure 7. The analysis of radioactivity uptake and immunohistochemistry in tumor tissue.

The average radioactivity uptake at 30 minutes corresponding to SPECT images (a) and the abundant NIS protein detected by immunohistochemistry (b) in Bac-NIS-SB-NeoR-U87 tumor tissue were performed. “*” = p < 0.05 (one-way ANOVA), Data = means ± SD (at least N = 3).