Document Type

Article

Publication Date

4-4-2025

Keywords

JMG, SS1, Animals, Integrases, Mice, Gene Editing, Recombination, Genetic, Mice, Inbred C57BL, Genome, Alleles

JAX Source

Genome Biol. 2025;26(1):85. Epub 20250404.

ISSN

1474-760X

PMID

40186303

DOI

https://doi.org/10.1186/s13059-025-03560-3

Grant

Research reported in this publication was partially supported by the National Cancer Institute under award numbers P30CA034196 and R01CA265978 (to V.H.) and R01DC015242 and R01DC018304 (to B.T.). The content is solely the respon- sibility of the authors and does not necessarily represent the official views of the NIH.

Abstract

BACKGROUND: The Cre-Lox system is a powerful tool in mouse genetics, enabling precise spatiotemporal control of gene expression and conditional knockout models. Since its development, it has transformed genome editing by facilitating targeted deletions, translocations, inversions, and complex modifications-double-floxed inverse orientation. Its utility extends beyond mice to rats, pigs, and zebrafish. However, challenges such as high costs, lengthy timelines, and unpredictable recombination remain, highlighting the need for ongoing improvements to enhance efficiency, reliability, and applicability across genetic models.

RESULTS: In this study, we perform a systematic analysis of Cre-mediated recombination in mice, creating 11 new strains with conditional alleles at the Rosa26 locus, using the C57BL/6J background. Factors influencing recombination efficiency include inter-loxP distance, mutant loxP sites, zygosity, chromosomal location, and breeder age. Our results demonstrate that the choice of Cre-driver strain plays a significant role in recombination efficiency. Optimal recombination is achieved when loxP sites are spaced by less than 4 kb and mutant loxP sites by 3 kb. Complete recombination fails with wildtype loxP sites spaced ≥ 15 kb or mutant lox71/66 sites spaced ≥ 7 kb. The best recombination efficiency is observed in breeders aged 8-20 weeks and when using heterozygous floxed alleles.

CONCLUSION: The Cre-Lox system remains indispensable for genetic engineering, offering flexibility beyond standalone applications by integrating with CRISPR-based methods to expand its utility. Despite challenges, our findings provide a framework for optimizing Cre-mediated recombination. By refining Cre-Lox strategies, this knowledge enhances experimental precision, improves reproducibility, and ultimately reduces the time and cost of genome modification.

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