PMID: 22333206Feb 16, 2012Paper

Construction and validation of dual fusion reporter gene expression vector for molecular imaging study

Zhonghua yi xue za zhi
Dan LiHong Shan

Abstract

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. 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. 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. EGFP-TfR dual fusion reporter gene expression vector has been successfully constructed, an...Continue Reading

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