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

Abstract

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 ( hTERT ) were critical for regulating its promoter in somatic cells. Accordingly, we engineered a humanized gene, hmTert , by knocking-in a 47-kilobase hybrid fragment containing these human non-coding sequences into the mTert locus in mouse embryonic stem cells (mESCs). The hmTert gene, encoding the wildtype mTert protein, was fully functional, as a mESC line with homozygous hmTert 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 hmTert 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.

Reverse transcriptase inhibition potentiates target therapy in BRAF-mutant melanomas: effects on cell proliferation, apoptosis, DNA-damage, ROS induction and mitochondrial membrane depolarization

“…Of note, similar findings have been previously observed in colorectal cancer [20]. Finally, an additional class of reverse transcriptase in eukaryotic cells is represented by telomerases (TERT), which are involved in the maintenance of chromosome ends [21]. In most adult human tissues, telomerase activity is low or undetectable.…”

Reverse transcriptase inhibition potentiates target therapy in BRAF-mutant melanomas: effects on cell proliferation, apoptosis, DNA-damage, ROS induction and mitochondrial membrane depolarization

“…Of note, similar ndings have been previously observed in colorectal cancer (20). Finally, an additional class of reverse transcriptase in eukaryotic cells is represented by telomerases (TERT), which are involved in the maintenance of chromosome ends (21). In most adult human tissues, telomerase activity is low or undetectable.…”

The regulatory function of tandem repeat VNTR2‐1 in hTERT gene involves basic Helix–loop–helix family transcription factors

“…MXD1 and MNT overexpression plasmids were generated by RT‐PCR. Total RNA was extracted from HEK293 cells and cDNAs were synthesized as previously described (Cheng et al, 2019). Primer pairs containing Flag tag coding sequence at 3′ end were used to amplify 705 bp MXD1 and 1088 bp MNT cDNAs.…”

“…For the analysis of endogenous hTERT transcription, HEK293 and HEK293‐∆VNTR2‐1 were transfected with TF overexpression plasmids and harvested 48 h post transfection. Total RNAs were extracted and qRT‐PCR assays were performed as previously described (Cheng et al, 2019). hTERT mRNA levels were normalized to that of GAPDH mRNA.…”