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.

Czech Centre for Phenogenomics Institute of Molecular Genetics of the Czech Academy of Sciences Prumyslova 595 252 50 Vestec Czech Republic

Institute of Biotechnology of the Czech Academy of Sciences BIOCEV 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

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Coleman, J. L. J. PubMed DOI PMC

Loulier, K. PubMed DOI

Skarnes, W. C. PubMed DOI PMC

Sket, T., Falcomatà, C. & Saur, D. Dual recombinase-based mouse models help decipher cancer biology and targets for therapy. PubMed DOI PMC

Kim, H., Kim, M., Im, S. K. & Fang, S. Mouse Cre-LoxP system: General principles to determine tissue-specific roles of target genes. PubMed DOI PMC

Karimova, M. PubMed DOI PMC

Karimova, M. PubMed DOI PMC

Kouvaros, S. et al. A CRE/DRE dual recombinase transgenic mouse reveals synaptic zinc-mediated thalamocortical neuromodulation. PubMed PMC

Wang, H. et al. Dual Cre and Dre recombinases mediate synchronized lineage tracing and cell subset ablation in vivo. PubMed PMC

Pettitt, S. J. PubMed DOI PMC

Jenickova, I. PubMed DOI

Chen, S. PubMed DOI PMC

Mizuno, N. PubMed DOI PMC

Kranz, A. PubMed DOI

Grieger, J. C., Choi, V. W. & Samulski, R. J. Production and characterization of adeno-associated viral vectors. PubMed DOI

Aurnhammer, C. PubMed DOI

Pandey, R. & Saluja, D. Hydrogen peroxide agarose gels for electrophoretic analysis of RNA. PubMed DOI

Luo, C. PubMed PMC

Takeo, T. & Nakagata, N. Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin. PubMed DOI

Deyle, D. R. & Russell, D. W. PubMed PMC

Wolter, J. M. PubMed DOI PMC

Grainge, I. & Jayaram, M. The integrase family of recombinases: Organization and function of the active site. PubMed DOI

Ludwig, D. L., Stringer, J. R., Wight, D. C., Doetschman, T. C. & Duffy, J. J. FLP-mediated site-specific recombination in microinjected murine zygotes. PubMed DOI

Wu, Y., Wang, C., Sun, H., LeRoith, D. & Yakar, S. High-efficient FLPo deleter mice in C57BL/6J background. PubMed DOI PMC

Luckow, B. PubMed DOI

Collins, D. E., Reuter, J. D., Rush, H. G. & Villano, J. S. Viral vector biosafety in laboratory animal research. PubMed PMC