Genetic Engineering of Humanized Telomere Mice

Telomere shortening is a hallmark of human aging, and telomerase regulation plays a critical role in cellular proliferation and replicative senescence. In human cells, telomere length imposes a limit on proliferative potential, a phenomenon known as the Hayflick limit. However, species-specific differences in telomere dynamics and telomerase regulation between humans and mice present challenges to using mice as accurate models for human telomere-related research. To address this limitation, we engineered a humanized telomerase gene (hmTert) in mice by replacing the non-coding sequences within the mouse Tert locus (mTert) with corresponding regulatory sequences from the human TERT gene. Breeding of these genetically modified mice resulted in progressive telomere shortening over successive generations, ultimately reaching human-like lengths (below 10 kb). This protocol outlines the development of this humanized telomere mouse model, referred to as HuT mice, offering a robust platform for studying human telomere biology and aging-related diseases. Key features • This protocol describes methods to increase the success rates of knocking in large genomic fragments (~47 kb) by integrating CRISPR-Cas9 with homologous recombination. • It enables precise engineering of a humanized telomerase gene (hmTert), faithfully recapitulating human TERT regulation and telomere length dynamics in mice.