无后续清理步骤的基因治疗微环生产方案。

IF 1.3 Q4 BIOCHEMICAL RESEARCH METHODS

STAR Protocols Pub Date : 2025-07-24 DOI:10.1016/j.xpro.2025.103982

Jonathan Do, Gautam Verma, Yasmin Maurice, Muniba Abdumanobova, Zicheng Deng, Tanya V Kalin, Vladimir V Kalinichenko

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引用次数: 0

摘要

目前生产微环的技术产量很低，因为需要清理步骤。在这里，我们提出了一种方案，通过消除诱导后的清理步骤，可以将微环产率提高10倍。我们描述了细菌培养、当日阿拉伯糖诱导和DNA收集的步骤。该方案适用于所有由含有attB， attP的亲本质粒产生的小环，以及在产生小环的细菌ZYCY10PS3T2中32个拷贝的ise - 1内切酶位点。

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Protocol for minicircle production for gene therapy without subsequent cleanup steps.

Current techniques for producing minicircles have low yields because of the cleanup steps required. Here, we present a protocol that can increase minicircle yield up to 10-fold by eliminating post-induction cleanup steps. We describe steps for bacterial culture, same-day arabinose induction, and DNA harvesting. This protocol is relevant for all minicircles produced from parental plasmids containing attB, attP, and 32 copies of Isce-I endonuclease sites in the minicircle-producing bacteria, ZYCY10PS3T2.

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