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 (<i>hmTert</i>) in mice by replacing the non-coding sequences within the mouse <i>Tert</i> locus (<i>mTert</i>) with corresponding regulatory sequences from the human <i>TERT</i> 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 (<i>hmTert</i>), faithfully recapitulating human TERT regulation and telomere length dynamics in mice.