title: [Construction and validation of dual fusion reporter gene expression vector for molecular imaging study].
abstract: OBJECTIVE
  To construct dual fusion reporter gene expression vector containing enhanced green fluorescence protein (EGFP) and human transferrin receptor (TfR), and validate the reconstructed plasmid, which will provide experimental foundation for in vivo dual-modality optical/Magnetic Resonance (MR) imaging.
  
  
  METHODS
  Clone TfR into the pEGFP-C1 vector to construct pEGFP-C1-TfR plasmid.pEGFP-C1-TfR plasmid was transfected into 293T cells for 48 h, then investigate EGFP expression under a fluorescence microscope; detect TfR expression through PT-PCR; inspect the subcellular location of EGFP-TfR fusion protein through Confocal Scanning Laser Microscopy; evaluate the function of EGFP-TfR fusion protein through Tf probe uptake and competition assays.
  
  
  RESULTS
  DNA sequencing analysis confirmed that EGFP-TfR gene sequence was correct, and there was no mutation and deletion. After transfecting the reconstructed plasmid into 293T cells, fluorescence microscope observation and RT-PCR results demonstrated that EGFP and TfR were expressed efficiently. EGFP-TfR fusion protein was located predominantly in the cellular membrane, and could specifically mediate internalization of Tf.
  
  
  CONCLUSIONS
  EGFP-TfR dual fusion reporter gene expression vector has been successfully constructed, and could be expressed efficiently with functional features. Thus, the expression vector could be applied for in vivo dual-modality optical/Magnetic Resonance (MR) imaging.
authors.affiliation: Laboratory of Molecular Imaging, Department of Radiology, the Third Affiliated Hospital of Sun Yat-sen University; Interventional Radiology Institute, Sun Yat-sen University, Guangzhou 510630, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
authors.forename: Jie
authors.initials: J
authors.lastname: Qin
authors.forename: Kang-shun
authors.initials: KS
authors.lastname: Zhu
authors.forename: Ming-sheng
authors.initials: MS
authors.lastname: Huang
authors.forename: Xiao-chun
authors.initials: XC
authors.lastname: Meng
authors.forename: Hong
authors.initials: H
authors.lastname: Shan
mesh_terms: D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008279:Magnetic Resonance Imaging / Q000379:methods*; D057054:Molecular Imaging; D010957:Plasmids; D011990:Receptors, Transferrin / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014162:Transfection
chemical_list: D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins