Conditionally immortalized retinal capillary endothelial cell lines (TR-iBRB) expressing differentiated endothelial cell functions derived from a transgenic rat

K. I. Hosoya, Masatoshi Tomi, S. Ohtsuki, H. Takanaga, M. Ueda, N. Yanai, M. Obinata, T. Terasaki

Research output: Contribution to journalArticle

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Abstract

The objective of this study was to establish and characterize a retinal capillary endothelial cell line (TR-iBRB) from a newly developed transgenic rat harboring the temperature-sensitive simian virus 40 (SV 40) large T-antigen gene (Tg rat). Retinal capillary endothelial cells were isolated from a Tg rat and cultured in collagen-coated dishes at 37°C for a period of 48 hr. Cells were subsequently cultured at 33°C to activate the large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells. Nine immortalized cell lines of retinal capillary endothelial cells (TR-iBRB1 ∼9) were obtained from a Tg rat. These cell lines had a spindle-fiber shape morphology, expressed the typical endothelial marker, von Willebrand factor, and internalized acetylated-low density lipoprotein. Moreover, vascular endothelial growth factor (VEGF) receptor-2 was expressed in TR-iBRBs. TR-iBRBs expressed a large T-antigen and grew well at 33°C with a doubling time of 19-21 hr. In contrast, cells did not grow at 37 and 39°C due to the reduced expression of large T-antigen. supporting temperature-dependent cell growth, TR-iBRBs expressed GLUT1 and exhibited 3-O-methyl-D-glucose (3-OMG) uptake activity, This 3-OMG uptake was saturable with a Michaelis-Menten constant of 5.56 ± 0.51 mM and a maximum uptake rate of 45.3 ± 2.6 nmol min-1 mg protein-1, P-Glycoprotein, with a molecular weight of ∼180 KDa, was expressed in TR-iBRBs. In addition, mdr 1a, mdr 1b and mdr 2 were detected in TR-iBRB2 using RT-PCR. In conclusion, conditionally immortalized retinal capillary endothelial cell lines were established from a transgenic rat harboring the temperature-sensitive SV 40 large T-antigen gene and these lines were shown to exhibit the properties of retinal capillary endothelial cells.

Original languageEnglish
Pages (from-to)163-172
Number of pages10
JournalExperimental Eye Research
Volume72
Issue number2
DOIs
Publication statusPublished - 2001
Externally publishedYes

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Transgenic Rats
Viral Tumor Antigens
Endothelial Cells
Cell Line
3-O-Methylglucose
Simian virus 40
Temperature
Vascular Endothelial Growth Factor Receptor-2
von Willebrand Factor
Genes
Collagen
Molecular Weight
Polymerase Chain Reaction
Growth
Proteins

Keywords

  • Blood-retinal barrier
  • GLUT1
  • P-glycoprotein
  • Retinal capillary endothelial cell line
  • SV 40 large T-antigen gene
  • Temperature-dependent cell growth
  • Vascular endothelial growth factor (VEGF) receptor-2

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Conditionally immortalized retinal capillary endothelial cell lines (TR-iBRB) expressing differentiated endothelial cell functions derived from a transgenic rat. / Hosoya, K. I.; Tomi, Masatoshi; Ohtsuki, S.; Takanaga, H.; Ueda, M.; Yanai, N.; Obinata, M.; Terasaki, T.

In: Experimental Eye Research, Vol. 72, No. 2, 2001, p. 163-172.

Research output: Contribution to journalArticle

Hosoya, K. I. ; Tomi, Masatoshi ; Ohtsuki, S. ; Takanaga, H. ; Ueda, M. ; Yanai, N. ; Obinata, M. ; Terasaki, T. / Conditionally immortalized retinal capillary endothelial cell lines (TR-iBRB) expressing differentiated endothelial cell functions derived from a transgenic rat. In: Experimental Eye Research. 2001 ; Vol. 72, No. 2. pp. 163-172.
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AU - Hosoya, K. I.

AU - Tomi, Masatoshi

AU - Ohtsuki, S.

AU - Takanaga, H.

AU - Ueda, M.

AU - Yanai, N.

AU - Obinata, M.

AU - Terasaki, T.

PY - 2001

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N2 - The objective of this study was to establish and characterize a retinal capillary endothelial cell line (TR-iBRB) from a newly developed transgenic rat harboring the temperature-sensitive simian virus 40 (SV 40) large T-antigen gene (Tg rat). Retinal capillary endothelial cells were isolated from a Tg rat and cultured in collagen-coated dishes at 37°C for a period of 48 hr. Cells were subsequently cultured at 33°C to activate the large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells. Nine immortalized cell lines of retinal capillary endothelial cells (TR-iBRB1 ∼9) were obtained from a Tg rat. These cell lines had a spindle-fiber shape morphology, expressed the typical endothelial marker, von Willebrand factor, and internalized acetylated-low density lipoprotein. Moreover, vascular endothelial growth factor (VEGF) receptor-2 was expressed in TR-iBRBs. TR-iBRBs expressed a large T-antigen and grew well at 33°C with a doubling time of 19-21 hr. In contrast, cells did not grow at 37 and 39°C due to the reduced expression of large T-antigen. supporting temperature-dependent cell growth, TR-iBRBs expressed GLUT1 and exhibited 3-O-methyl-D-glucose (3-OMG) uptake activity, This 3-OMG uptake was saturable with a Michaelis-Menten constant of 5.56 ± 0.51 mM and a maximum uptake rate of 45.3 ± 2.6 nmol min-1 mg protein-1, P-Glycoprotein, with a molecular weight of ∼180 KDa, was expressed in TR-iBRBs. In addition, mdr 1a, mdr 1b and mdr 2 were detected in TR-iBRB2 using RT-PCR. In conclusion, conditionally immortalized retinal capillary endothelial cell lines were established from a transgenic rat harboring the temperature-sensitive SV 40 large T-antigen gene and these lines were shown to exhibit the properties of retinal capillary endothelial cells.

AB - The objective of this study was to establish and characterize a retinal capillary endothelial cell line (TR-iBRB) from a newly developed transgenic rat harboring the temperature-sensitive simian virus 40 (SV 40) large T-antigen gene (Tg rat). Retinal capillary endothelial cells were isolated from a Tg rat and cultured in collagen-coated dishes at 37°C for a period of 48 hr. Cells were subsequently cultured at 33°C to activate the large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells. Nine immortalized cell lines of retinal capillary endothelial cells (TR-iBRB1 ∼9) were obtained from a Tg rat. These cell lines had a spindle-fiber shape morphology, expressed the typical endothelial marker, von Willebrand factor, and internalized acetylated-low density lipoprotein. Moreover, vascular endothelial growth factor (VEGF) receptor-2 was expressed in TR-iBRBs. TR-iBRBs expressed a large T-antigen and grew well at 33°C with a doubling time of 19-21 hr. In contrast, cells did not grow at 37 and 39°C due to the reduced expression of large T-antigen. supporting temperature-dependent cell growth, TR-iBRBs expressed GLUT1 and exhibited 3-O-methyl-D-glucose (3-OMG) uptake activity, This 3-OMG uptake was saturable with a Michaelis-Menten constant of 5.56 ± 0.51 mM and a maximum uptake rate of 45.3 ± 2.6 nmol min-1 mg protein-1, P-Glycoprotein, with a molecular weight of ∼180 KDa, was expressed in TR-iBRBs. In addition, mdr 1a, mdr 1b and mdr 2 were detected in TR-iBRB2 using RT-PCR. In conclusion, conditionally immortalized retinal capillary endothelial cell lines were established from a transgenic rat harboring the temperature-sensitive SV 40 large T-antigen gene and these lines were shown to exhibit the properties of retinal capillary endothelial cells.

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