The experimental study of monitoring Stem Cells using a baculovirus vector to deliver reporter gene

来源 :The 1st Sino-American Conference on Nuclear Medicine(首届中美核医学 | 被引量 : 0次 | 上传用户:wangyizhinihao
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  Objective: To investigate the feasibility of monitoring stem cells using baculovirus vectors for the delivery of the sodium-iodide symporter (NIS) gene as a reporter.Methods: Induced pluripotent stem cells (iPSC), human embryonic stem cells (hESC), bone marrow mesenchymal stem cells (BMSC), were infected with recombinant green fluorescent protein baculovirus (Bac-GFP) at different multiplicities of infection (MOIs).The infection efficiency and the longevity of gene expression were determined by flow cytometry (FCM).An in vitro cytotoxicity assay was also conducted after infection of stem cells with Bac-NIS.Stem cells were transduced by recombinant baculovirus expressing NIS (Bac-NIS) and NIS protein expression was detected by Westen Blot.The iodine uptake at different time by stem cells infected with Bac-NIS and inhibition of this uptake by sodium perchlorate were measured using a γ counter.Results: We have successfully constructed recombinant baculoviruses carrying NIS and GFP under the control of the cytomegalovirus promoter.Bac-GFP infects BMSC at an efficiency of 80% with MOI of 100, and the infection efficiency of stem cells increases with the MOI; and also there are high infection efficiency for iPSC and hESC with Bac-GFP.There is no difference about the mortality of stem cells at the MOI of 400 between the positive group and the control group within 6 days.About 80% and 60% of stem cells were positive GFP at the day of 7 and 14,respectively.BMSC infected with Bac-NIS expressed NIS protein, the expression of NIS had a capability of concentrating iodide and the peak for the iodine intake was about 30min, which could be inhibited by sodium perchlorate.Conclusion: The recombinant NIS gene baculovirus reporting system can effectively carry out the monitoring of stem cells, and no cell toxicity.
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