Conditionally immortalized retinal capillary endothelial cell lines (TR-iBRB) expressing differentiated endothelial cell functions derived from a transgenic rat.
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 degrees C for a period of 48 hr. Cells were subsequently cultured at 33 degrees 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 approximately 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 degrees C with a doubling time of 19-21 hr. In contrast, cells did not grow at 37 and 39 degrees 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 m M and a maximum uptake rate of 45.3 +/- 2.6 nmol min(-1) mg protein(-1). P-Glycoprotein, with a molecular weight of approximately 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.
Hosoya
K
K
Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Sendai, Japan.
Tomi
M
M
Ohtsuki
S
S
Takanaga
H
H
Ueda
M
M
Yanai
N
N
Obinata
M
M
Terasaki
T
T
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis; D000818:Animals; D030801:Animals, Genetically Modified; D000957:Antigens, Viral, Tumor / Q000032:analysis / Q000235:genetics; D001813:Blood-Retinal Barrier; D015153:Blotting, Western; D002196:Capillaries; D002455:Cell Division; D002469:Cell Separation; D004730:Endothelium, Vascular / Q000473:pathology*; D019450:Genes, MDR; D051272:Glucose Transporter Type 1; D006358:Hot Temperature; D023421:Models, Animal; D009004:Monosaccharide Transport Proteins / Q000032:analysis; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis; D017978:Receptors, Growth Factor / Q000032:analysis; D040262:Receptors, Vascular Endothelial Growth Factor; D012171:Retinal Vessels; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000957:Antigens, Viral, Tumor; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D017978:Receptors, Growth Factor; C495679:Slc2a1 protein, rat; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor