TY - JOUR
T1 - Cationic polymer-mediated genetic transduction into cultured human chondrosarcoma-derived HCS-2/8 cells
AU - Ohashi, Suzuyo
AU - Kubo, Toshikazu
AU - Ikeda, Takumi
AU - Arai, Yuji
AU - Takahashi, Kenji
AU - Hirasawa, Yasusuke
AU - Takigawa, Masaharu
AU - Satoh, Etsuko
AU - Imanishi, Jiro
AU - Mazda, Osam
N1 - Funding Information:
Acknowledgments. This study was supported in part by grants from the Hip Joint Foundation of Japan, and by a Grant-in-Aid for Scientific Research (No. 11470314) from the Ministry of Education, Science, Sports, and Culture of Japan.
PY - 2001
Y1 - 2001
N2 - The usefulness of three types of cationic polymer, i.e., degraded polyamidoamine (PAMAM) dendrimer (SuperFect Transfection Reagent; Qiagen), linear polyethylenimine (PEI: ExGen 500: Euromedex), and branched PEI in gene delivery into chondrocytes was examined comparatively. A plasmid vector containing the Escherichia coli LacZ (pSES.β) was combined with one of the three cationic polymers at various molar ratios and the resultant complex (polyplex) was used to transduce a human chondrocyte-like cell line, HCS-2/8. Gene expression was evaluated by an O-nitrophenyl β-D-galactopyranoside (ONPG) assay and by staining with 0.05% 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal: Nacalai Tesque). The ONPG assay showed that the highest delivery rate was achieved when 2μg of pSES.β was combined with either 21 μg of dendrimer, 1.7 μg of linear PEI, or 2.0 μg of branched PEI. At the same DNA/polymer ratios, the proportions of X-galstained cells were also the highest (31.3 ± 7.5%, 30.3 ± 9.0%, and 8.3 ± 3.1%, respectively). LacZ expression reached the highest level 3 days after the dendrimer-mediated transduction, and gradually declined, returning to the background level on day 14. Possible cytotoxicity was examined by trypan blue staining and phase contrast microscopic observations. Neither cytotoxicity nor morphological change was induced at the optimal dose of each polymer. The cationic polymers, particularly the degraded dendrimer and linear PEI, would be a useful nonviral vector for gene delivery to cells of chondrocytes.
AB - The usefulness of three types of cationic polymer, i.e., degraded polyamidoamine (PAMAM) dendrimer (SuperFect Transfection Reagent; Qiagen), linear polyethylenimine (PEI: ExGen 500: Euromedex), and branched PEI in gene delivery into chondrocytes was examined comparatively. A plasmid vector containing the Escherichia coli LacZ (pSES.β) was combined with one of the three cationic polymers at various molar ratios and the resultant complex (polyplex) was used to transduce a human chondrocyte-like cell line, HCS-2/8. Gene expression was evaluated by an O-nitrophenyl β-D-galactopyranoside (ONPG) assay and by staining with 0.05% 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal: Nacalai Tesque). The ONPG assay showed that the highest delivery rate was achieved when 2μg of pSES.β was combined with either 21 μg of dendrimer, 1.7 μg of linear PEI, or 2.0 μg of branched PEI. At the same DNA/polymer ratios, the proportions of X-galstained cells were also the highest (31.3 ± 7.5%, 30.3 ± 9.0%, and 8.3 ± 3.1%, respectively). LacZ expression reached the highest level 3 days after the dendrimer-mediated transduction, and gradually declined, returning to the background level on day 14. Possible cytotoxicity was examined by trypan blue staining and phase contrast microscopic observations. Neither cytotoxicity nor morphological change was induced at the optimal dose of each polymer. The cationic polymers, particularly the degraded dendrimer and linear PEI, would be a useful nonviral vector for gene delivery to cells of chondrocytes.
KW - Branched polyethylenimine
KW - Chondrocytes
KW - Degraded polyamidoamine dendrimer
KW - Gene delivery
KW - Linear polyethylenimine
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U2 - 10.1007/s007760170028
DO - 10.1007/s007760170028
M3 - Article
C2 - 11289590
AN - SCOPUS:0035123266
VL - 6
SP - 75
EP - 81
JO - Journal of Orthopaedic Science
JF - Journal of Orthopaedic Science
SN - 0949-2658
IS - 1
ER -