位点特异性重组酶(SSR)对于实现工程等位基因的精确时空控制至关重要。这些酶在促进侧翼为重组位点的基因座的缺失或倒位中起关键作用。导致内源性基因的激活或抑制,选择标记或报告元素。然而,复杂等位基因中的多重重组可能是费力的。为了解决这个问题,已经开发了一种使用AAV载体的新的有效方法,以简化基于Cre的系统转换,FLP,Dre和Vika重组酶。在这项研究中,我们提出了一种使用Cre进行离体等位基因转换的有效方法,FLP(翻转),德雷,和维卡重组酶,使用腺相关病毒(AAV)作为递送载体。AAV能够在报道小鼠系中以最小的毒性进行有效的等位基因转化。此外,AAV促进序列等位基因转换,对于完全转换具有多个重组位点的等位基因至关重要,通常在条件敲除小鼠模型中发现。虽然简单的等位基因转化显示出100%的效率,复杂的多重转换始终实现80%的转换率。总的来说,这种策略显着减少了对动物的需求,并显着加快了等位基因转化的过程,代表了基因组工程技术的重大改进。
Site-specific recombinases (SSRs) are critical for achieving precise spatiotemporal control of engineered alleles. These enzymes play a key role in facilitating the deletion or inversion of loci flanked by recombination sites, resulting in the activation or repression of endogenous genes, selection markers or reporter elements. However, multiple recombination in complex alleles can be laborious. To address this, a new and efficient method using AAV vectors has been developed to simplify the conversion of systems based on Cre, FLP, Dre and Vika recombinases. In this study, we present an effective method for ex vivo allele conversion using Cre, FLP (flippase), Dre, and Vika recombinases, employing adeno-associated viruses (AAV) as delivery vectors. AAVs enable efficient allele conversion with minimal toxicity in a reporter mouse line. Moreover, AAVs facilitate sequential allele conversion, essential for fully converting alleles with multiple recombination sites, typically found in conditional knockout mouse models. While simple allele conversions show a 100% efficiency rate, complex multiple conversions consistently achieve an 80% conversion rate. Overall, this strategy markedly reduces the need for animals and significantly speeds up the process of allele conversion, representing a significant improvement in genome engineering techniques.