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Endocytosis of a functionally enhanced GFP-tagged transferrin receptor in CHO cells.

Abstract
The endocytosis of transferrin receptor (TfR) has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP)-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81 ± 3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.
AuthorsQi He, Xiaoxu Sun, Chong Chu, Qing Jiang, Huifen Zhu, Yong He, Tingting Yue, Ruibo Wang, Ping Lei, Guanxin Shen
JournalPloS one (PLoS One) Vol. 10 Issue 3 Pg. e0122452 ( 2015) ISSN: 1932-6203 [Electronic] United States
PMID25803700 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Antibodies, Monoclonal
  • DNA Primers
  • Receptors, Transferrin
  • Recombinant Fusion Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
Topics
  • Animals
  • Antibodies, Monoclonal (metabolism)
  • Blotting, Western
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • DNA Primers (genetics)
  • Endocytosis (physiology)
  • Flow Cytometry
  • Fluorescent Antibody Technique
  • Green Fluorescent Proteins (metabolism)
  • Humans
  • Immunohistochemistry
  • Nanoparticles (metabolism)
  • Plasmids (genetics)
  • Receptors, Transferrin (metabolism, physiology)
  • Recombinant Fusion Proteins (metabolism)
  • Statistics, Nonparametric

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