Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prumyslova 595, 252 50, Vestec, Czech Republic.
Laboratory of Transgenic Models of Diseases, Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 142 20, Prague, Czech Republic.
Sci Rep. 2024 Aug 29;14(1):20160. doi: 10.1038/s41598-024-70853-1.
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.
位点特异性重组酶（SSRs）对于实现工程化等位基因的精确时空控制至关重要。这些酶在促进由重组位点侧翼的基因座缺失或倒位方面发挥着关键作用，从而激活或抑制内源性基因、选择标记或报告元件。然而，在复杂等位基因中进行多次重组可能很繁琐。为了解决这个问题，已经开发了一种新的、高效的使用 AAV 载体的方法，以简化基于 Cre、FLP、Dre 和 Vika 重组酶的系统的转换。在这项研究中，我们提出了一种使用 Cre、FLP（翻转酶）、Dre 和 Vika 重组酶进行体外等位基因转换的有效方法，使用腺相关病毒（AAV）作为递送载体。AAV 能够以最小的毒性在报告小鼠品系中实现高效的等位基因转换。此外，AAV 促进了顺序等位基因转换，这对于完全转换具有多个重组位点的等位基因至关重要，这些重组位点通常存在于条件性敲除小鼠模型中。虽然简单的等位基因转换显示出 100%的效率，但复杂的多次转换始终实现 80%的转换率。总体而言，该策略大大减少了对动物的需求，并显著加快了等位基因转换的过程，代表了基因组工程技术的重大改进。
