Cationic polymer-mediated genetic transduction into cultured human chondrosarcoma-derived HCS-2/8 cells.
The usefulness of three types of cationic polymer, i.e., degraded polyamidoamine (PAMAM) dendrimer (SuperFect Transfection Reagent; Oiagen), 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.beta) 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 beta-D-galactopyranoside (ONPG) assay and by staining with 0.05% 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal; Nacalai Tesque). The ONPG assay showed that the highest delivery rate was achieved when 2microg of pSES.beta was combined with either 21 microg of dendrimer, 1.7microg of linear PEI, or 2.0microg of branched PEI. At the same DNA/polymer ratios, the proportions of X-gal-stained 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.
Ohashi
S
S
Department of Orthopaedic Surgery, Kyoto Prefectural University of Medicine, Japan.
Kubo
T
T
Ikeda
T
T
Arai
Y
Y
Takahashi
K
K
Hirasawa
Y
Y
Takigawa
M
M
Satoh
E
E
Imanishi
J
J
Mazda
O
O
D001859:Bone Neoplasms / Q000235:genetics; D019902:Chondrocytes; D002813:Chondrosarcoma / Q000235:genetics; D050091:Dendrimers; D015870:Gene Expression; D006801:Humans; D010957:Plasmids; D011073:Polyamines; D011094:Polyethyleneimine; D014161:Transduction, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D050091:Dendrimers; C104700:PAMAM Starburst; D011073:Polyamines; D011094:Polyethyleneimine