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Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo
被引:143
作者:
Chen, ZY
He, CY
Kay, MA
机构:
[1] Stanford Univ, Sch Med, Dept Pediat, Stanford, CA 94305 USA
[2] Stanford Univ, Sch Med, Dept Genet, Stanford, CA 94305 USA
关键词:
D O I:
10.1089/hum.2005.16.126
中图分类号:
Q81 [生物工程学(生物技术)];
Q93 [微生物学];
学科分类号:
071005 ;
0836 ;
090102 ;
100705 ;
摘要:
We have shown previously that minicircle DNA vectors free of plasmid bacterial DNA sequences are capable of persistent high level of transgene expression in vivo. The minicircle is generated in bacteria from a parental plasmid containing an inducible phage phiC31 integrase gene and a therapeutic expression cassette flanked with attB and attP sites. The phiC31-mediated intramolecular recombination between attB and attP results in the formation of two circular DNA molecules, one containing the eukaryotic expression cassette ( minicircle), and the other the plasmid bacterial DNA backbone ( BB). Previously, the minicircle was purified away from the plasmid BB by a restriction enzyme digestion step and ultracentrifugation in cesium chloride. We have now included the endonuclease I-SceI gene together with its recognition site in the minicircle-producing plasmid to allow the linearization and degradation of the plasmid BB in bacteria. The minicircle can then be isolated by routine plasmid purification procedures such as a one-step affinity column. With additional modifications to our previous strategy, we can prepare a minicircle encoding a 4-kb human factor IX expression cassette, up to 1.8 mg of minicircle with 97% purity was prepared from a 1 liter bacterial culture. The high yield, simple purification, and robust and persistent transgene expression make these vectors viable for gene therapy applications.
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页码:126 / 131
页数:6
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