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

He Q, Sun X, Chu C, Jiang Q, Zhu H, He Y, Yue T, Wang R, Lei P, Shen G - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.

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.

No MeSH data available.


Related in: MedlinePlus

Construction and Expression of hTfR-EGFP in CHO cells.(A) Sal I and BglII restriction enzyme digestion analysis. M: 1kb DNA ladder; lane 1: pEGFP-hTfR, lane 2: pGEM-T-hTfR, lane 3: pEGFP-C1. (B) Immunoblot analysis of hTfR expression in cells. Cell lysates were probed by mouse anti-human TfR mAb. Left: representative WB picture of 4 separate experiments was shown. Right: Densitometric analysis of hTfR levels of the western blots. P values were calculated on the basis of Dunnett-t test (NS, not significant). (C) EGFP expression in CHO cells was detected by FCM and fluorescence microscope (insert).
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pone.0122452.g002: Construction and Expression of hTfR-EGFP in CHO cells.(A) Sal I and BglII restriction enzyme digestion analysis. M: 1kb DNA ladder; lane 1: pEGFP-hTfR, lane 2: pGEM-T-hTfR, lane 3: pEGFP-C1. (B) Immunoblot analysis of hTfR expression in cells. Cell lysates were probed by mouse anti-human TfR mAb. Left: representative WB picture of 4 separate experiments was shown. Right: Densitometric analysis of hTfR levels of the western blots. P values were calculated on the basis of Dunnett-t test (NS, not significant). (C) EGFP expression in CHO cells was detected by FCM and fluorescence microscope (insert).

Mentions: To prepare fluorescent- labeled hTfR, the EGFP was fused to the amino terminus of full-length hTfR (Fig 1). Restriction enzyme digestion (Fig 2A) and DNA sequencing (data not shown) confirmed that hTfR cDNA had been successfully cloned into pEGFP-C1 and the predicted amino acid sequence of hTfR were in agreement with NM_003234.2 and NP_003225.2 in GenBank database and published reports [12,13].


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

He Q, Sun X, Chu C, Jiang Q, Zhu H, He Y, Yue T, Wang R, Lei P, Shen G - PLoS ONE (2015)

Construction and Expression of hTfR-EGFP in CHO cells.(A) Sal I and BglII restriction enzyme digestion analysis. M: 1kb DNA ladder; lane 1: pEGFP-hTfR, lane 2: pGEM-T-hTfR, lane 3: pEGFP-C1. (B) Immunoblot analysis of hTfR expression in cells. Cell lysates were probed by mouse anti-human TfR mAb. Left: representative WB picture of 4 separate experiments was shown. Right: Densitometric analysis of hTfR levels of the western blots. P values were calculated on the basis of Dunnett-t test (NS, not significant). (C) EGFP expression in CHO cells was detected by FCM and fluorescence microscope (insert).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4372551&req=5

pone.0122452.g002: Construction and Expression of hTfR-EGFP in CHO cells.(A) Sal I and BglII restriction enzyme digestion analysis. M: 1kb DNA ladder; lane 1: pEGFP-hTfR, lane 2: pGEM-T-hTfR, lane 3: pEGFP-C1. (B) Immunoblot analysis of hTfR expression in cells. Cell lysates were probed by mouse anti-human TfR mAb. Left: representative WB picture of 4 separate experiments was shown. Right: Densitometric analysis of hTfR levels of the western blots. P values were calculated on the basis of Dunnett-t test (NS, not significant). (C) EGFP expression in CHO cells was detected by FCM and fluorescence microscope (insert).
Mentions: To prepare fluorescent- labeled hTfR, the EGFP was fused to the amino terminus of full-length hTfR (Fig 1). Restriction enzyme digestion (Fig 2A) and DNA sequencing (data not shown) confirmed that hTfR cDNA had been successfully cloned into pEGFP-C1 and the predicted amino acid sequence of hTfR were in agreement with NM_003234.2 and NP_003225.2 in GenBank database and published reports [12,13].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.

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.

No MeSH data available.


Related in: MedlinePlus