Engineering a humanized telomerase reverse transcriptase gene in mouse embryonic stem cells

<jats:title>Abstract</jats:title><jats:p>Telomerase is expressed in adult mouse, but not in most human, tissues and mouse telomeres are much longer than those in humans. This interspecies difference of telomere homeostasis poses a challenge in modeling human diseases using laboratory mice. Using chromatinized bacterial artificial chromosome reporters, we discovered that the 5′ intergenic region, introns 2 and 6 of human telomerase gene (<jats:italic>hTERT</jats:italic>) were critical for regulating its promoter in somatic cells. Accordingly, we engineered a humanized gene, <jats:italic>hmTert</jats:italic>, by knocking-in a 47-kilobase hybrid fragment containing these human non-coding sequences into the <jats:italic>mTert</jats:italic> locus in mouse embryonic stem cells (mESCs). The <jats:italic>hmTert</jats:italic> gene, encoding the wildtype mTert protein, was fully functional, as a mESC line with homozygous <jats:italic>hmTert</jats:italic> alleles proliferated for over 400 population doublings without exhibiting chromosomal abnormalities. Like human ESCs, the engineered mESCs contained high telomerase activity, which was repressed upon their differentiation into fibroblast-like cells in a histone deacetylase-dependent manner. Fibroblast-like cells differentiated from these mESCs contained little telomerase activity. Thus, telomerase in mESCs with the <jats:italic>hmTert</jats:italic> alleles was subjected to human-like regulation. Our study revealed a novel approach to engineer a humanized telomerase gene in mice, achieving a milestone in creating a mouse model with humanized telomere homeostasis.</jats:p>