Cre-LoxP条件性基因敲除的实际应用策略

Practical Application Strategies of Cre-LoxP Conditional Gene Knockout

Cre-loxP系统是起源于P1噬菌体的高效重组系统,其根据loxP位点组合不同而发生特定重组的模式使其成为近年来基因编辑最常用的工具之一。本文聚焦于Cre-loxP系统的实际应用问题,首先分析了CRISPR/Cas9系统在插入Cre序列与loxP序列中的作用与优势,随后阐述了一系列Cre-loxP系统的实际应用问题。本文阐述了Cre重组酶序列的原位插入与安全位点插入的选用、loxP序列插入的策略、Cre重组酶的标签蛋白鉴定、“异位”表达的荧光鉴定、PCR鉴定法的引物设计与工具鼠的繁殖策略等。同时,介绍了Cre-loxP系统在条件性基因敲除中的优化应用,例如配体诱导型Cre、启动子激活型Cre、光诱导型Cre与活性改造型Cre等。通过这些优化应用,可以获得对条件性基因敲除的时间可控性,也可以调节Cre重组酶的活性,甚至可以规避Cre重组酶本身的毒性。最后,论述了Cre-loxP系统面临的缺陷与挑战,展望了未来Cre-loxP系统的发展方向。总而言之,本文综述了基于Cre-loxP系统的基因敲除的实际应用,总结了目前Cre-loxP系统最前沿的研究进展和优化策略,并对未来基于Cre-loxP系统的基因编辑进行展望。本文旨在提供解决基于Cre-loxP系统实际操作问题的理论指导,并为未来更精确、更可控、更具适应性的基因编辑提供新的研究思路。

Cre-loxP is an efficient recombination system originated from P1 phage.Its specific recombination patterns based on the locus of X-over P1 make it one of the most commonly used tools for gene editing in recent years.This paper focuses on the practical application of Cre-loxP system.Firstly,the functions and advantages of CRISPR/Cas9 system in Cre sequence insertion and loxP sequence insertion are analyzed.Then,a series of practical application problems of Cre-loxP system are described.For example,in this paper,the selection of Cre recombinase sequence in situ and safe site,the strategy of loxP sequence insertion,the identification of Cre recombinase tag protein,fluorescence identification of"ectopic"expression,primer design of PCR identification and reproductive strategy of mouse were described.At the same time,the optimization of Cre-loxP system in conditional gene knockout is introduced,such as ligand-induced Cre,promoter activated Cre,photo-induced Cre and activity modification of Cre.Through these optimized applications,we can obtain time-controlled conditional gene knockout,regulate the activity of Cre recombinase,and even avoid the toxicity of Cre recombinase itself.Finally,this paper discusses the defects and challenges of Cre-loxP system,and looks into the future development direction of Cre-loxP system.In summary,this paper reviews the practical application of gene knockout based on Cre-loxP system,summarizes the latest research progress and optimization strategies of Cre-loxP system,and prospects the future gene editing based on Cre-loxP system.This paper aims to provide theoretical guidance for solving practical operation problems based on Cre-loxP system,and to provide new research ideas for more accurate,more controllable and more adaptive gene editing in the future.