Pre-deliver chitosanase to cells: a novel strategy to improve gene expression by endocellular degradation-induced vector unpacking.
A radio-labeled plasmid pTracer/Bsd/LacZ containing LacZ reporter gene was complexed with different molecular weights of chitosans (CS). Mouse myoblast cell line C2C12 was transfected by these chitosan-plasmid DNA complexes, and lipofectamine 2000 was used as control. Forty-eight hours after transfection, the activity of beta-galactosidase and radioactive count of cell lysis were determined. It was found that chitosan, especially low molecular weight species, had a surprising ability to deliver DNA into cells, since the radioactive count of cells transfected by chitosan-DNA complexes was even two times that of cells transfected by lipofectamine 2000. But the beta-galactosidase activity of chitosan/DNA complexes was much lower compared to that of lipofectamine 2000. Chitosanase which could degrade chitosan in specific mode was transported into C2C12 cells by osmotic lysis prior to gene delivery. Then these chitosanase-modified cells were transfected by CS-DNA complexes. The results indicated that beta-galactosidase activity in these cells increased markedly to 425.4 +/- 45.1 U/mg protein, nearly two-fold as that of cells transfected by liposome. This transfection protocol was also applied to 3T3 mouse fibroblast, 2T3 mouse osteoblast and MG63 human osteosarcoma cell lines, and an increased gene expression level was observed without exception. It is thought that the incorporated chitosanase could aid in chitosan degradation, which would promote gene unpacking, consequently increasing gene expression.
Liang
Dong Chun
DC
Institute of Endocrinology, Tianjin Medical University, Tianjin 300070, China. liangdongchun1225@263.net
Liu
Wen Guang
WG
Zuo
Ai Jun
AJ
Sun
Shu Jun
SJ
Cheng
Nan
N
Guo
Gang
G
Zhang
Jing Yu
JY
De Yao
Kang
K
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D048271:Chitosan / Q000737:chemistry; D005347:Fibroblasts / Q000201:enzymology; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007763:Lac Operon; D008055:Lipids / Q000737:chemistry; D051379:Mice; D008970:Molecular Weight; D032446:Myoblasts / Q000201:enzymology; D010006:Osteoblasts / Q000201:enzymology; D010316:Particle Size; D010957:Plasmids; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis
D008055:Lipids; C086724:Lipofectamine; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase; D001616:beta-Galactosidase