Improved transfection efficiency of CS/DNA complex by co-transfected chitosanase gene.
Previously, we had demonstrated that insufficient intracellular unpacking of exogene from its chitosan carrier contributes towards the restricted transfection efficiency of CS/DNA complex. In order to enhance intracellular unpacking and thus improve the transfection efficiency, our present work has addressed a novel strategy of chitosanase gene (csn) co-transfection. An Aspergillus fumigatuscsn gene was semi-synthesized and cloned into a prokaryotic expression vector, plasmid pGEX-3X, meanwhile a mutant csn gene encoding an inactive Asp129-Asn chitosanase was generated by site-directed mutagenesis. Both active csn (acCSN) and inactive csn (inCSN) genes were expressed in bacteria cells and chitosan degradation activities of those purified recombinant proteins were tested. These csn genes were further subcloned into an eukaryotic expression vector, plasmid pTracer-CMV/Bsd, containing a gfp reporter gene. Recombinant plasmid pTracer-accsn or pTracer-incsn was co-transfected with plasmid pTracer/Bsd/LacZ, which contains an additional lacZ reporter gene, into C2C12 myoblast cells by CS/DNA complex. The expression of gfp reporter gene was determined by fluorescence microscope, while the expression of lacZ reporter was evaluated quantitatively by beta-galactosidase activity. All together, findings indicate that during the exogene being delivered into mammalian cells by CS/DNA complex, the csn co-transfection is beneficial for the exogene expression.
Zuo
Aijun
A
Tianjin Medical University, Institute of Endocrinology, Tianjin 300070, China.
Sun
Pei
P
Liang
Dongchun
D
Liu
Wenguang
W
Zhao
Ronglan
R
Guo
Gang
G
Cheng
Nan
N
Zhang
Jingyu
J
Yao
Kangde
K
D021581:Active Transport, Cell Nucleus; D000818:Animals; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D048271:Chitosan / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection / Q000379:methods*
D005656:Fungal Proteins; D011994:Recombinant Proteins; D004247:DNA; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase