Modification of the telomerase gene with human regulatory sequences resets mouse telomeres to human length

Abstract

Telomeres shorten with each cell division, serving as biomarkers of aging, with human tissues exhibiting short telomeres and restricted telomerase expression. In contrast, mice have longer telomeres and widespread telomerase activity, limiting their relevance as models for human telomere biology. To address this, we engineer a mouse strain with a humanized mTert gene (hmTert), replacing specific non-coding sequences with human counterparts. The hmTert gene, which is repressed in adult tissues except the gonads and thymus, closely mimics human TERT regulation. This modification rescues telomere dysfunction in mTert-knockout mice. Successive intercrosses of Tert^h/- mice stabilized telomere length below 10 kb, while Tert^h/h mice achieve a human-like average length of 10-12 kb, compared to 50 kb in wildtype mice. Despite shortened telomeres, Tert^h/h mice maintain normal body weight and cell homeostasis. These mice, with humanized telomere regulation, represent a valuable model to study human aging and cancer.

Fig. 1. The hmTert gene and its expression in mice.

a Genomic maps of hTERT, mTert, and hmTert loci. Arrows indicate the directions of transcription. Vertical lines are exons; black and dark grey regions represent repetitive sequences, TEs, and VNTRs, respectively. Human and mouse 5’IR and introns 2 & 6 are labeled in blue and red, respectively. b Telomerase expression in tissues from Tert^+/- and Tert^h/- littermates. Telomerase activities were determined by TRAP assay. 0.5 µg protein extracts from 4-month-old mice were used except for thymus (0.12 µg). +, h, and - refer to mTert, hmTert, and mTert-KO alleles, respectively. Organs from at least three different mice were examined and data from one representative mouse are shown here. c The expression of Tert mRNAs in adult mice. Tissues were collected from 4-month-old Tert^h/+ mice. hmTert and mTert mRNAs were distinguished by using primers overlapping with the silent mutations in exon 2 of the hmTert allele. P values, two tailed student’s t test, are shown. d hTERT mRNA expression in human tissues. Tert/TERT mRNA data were determined by qRT-PCR assay, normalized to 18S rRNA, and compared to those in Tert^h/+ ESCs or human ESC H1 cells (1.0). For c, d means and standard deviations (±SDs) of three technical repeats are shown. Repeated experiments showed similar results. Source data for panels b–d are provided as Source Data files.

Fig. 2. Functions of the hmTert gene in mice.

a Breeding strategy. Telomere length of splenocytes from 2-month-old Tert^+/-, Tert^h/-, and Tert^-/- mice were determined by Flow-FISH (b) and telomere restriction fragment (TRF) analysis (c). b Telomere Flow-FISH. Telomere signals were detected by hybridization to FAM-(CCCTAA)₃ oligonucleotide. Fluorescence signals were compared to that of wildtype C57BL/6 J mice (1.0). c TRF analysis. Splenocyte genomic DNAs were digested with HinfI and RsaI, followed by pulsed-field gel electrophoresis and Southern blotting. Positions of size markers are shown on the left (kb). d Litter sizes of breeding between Tert^+/- and Tert^-/- (red triangles), Tert^h/- and Tert^-/- (blue circles), Tert^-/- and Tert^-/- (white squares) mice. e Body weight of male (upper) and female (lower) mice at 8-week of age. f Testis weight of mice at 10–15-week age. g H&E staining of seminiferous tubules in testes from Tert^+/-, Tert^h/-, and Tert^-/- mice. Yellow arrowheads indicate aberrant tubules. h Average percentages of aberrant seminiferous tubules in testes. +/+, n = 3; +/-: G3, n = 3; G4, n = 2, G5, n = 3; h/-: G3, n = 3, G4, n = 3, G5, n = 4; -/-: G3, n = 3, G4, n = 3, G5, n = 3. i, j, Survival curves of mice with mTert, hmTert, and mTert-KO alleles. Mice were bred as shown in panel a. Kaplan-Meier survival curves of G4 (i) and G5 (j) mice are shown. P-values of survival curve comparisons were calculated using logrank test. Each datapoint in panels b, d, e, f, & h represents one biological repeat (one animal). Means ± SDs are indicated. Source data for panels b–f and h–j are provided as Source Data files.

Fig. 3. Comparisons of G6 mice with mTert and hmTert alleles.

a Mouse breeding scheme. b TRF analysis of representative animals. Splenocyte genomic DNAs were digested with HinfI and RsaI, followed by pulsed-field gel electrophoresis and Southern blotting. c Kaplan-Meier survival curves of mice. P-values comparing indicated paired curves were determined using logrank tests. d Body weight of male (upper) and female (lower) mice at 8-week of age. e Testis weight of mice at 10−15-week age. Each datapoint in panels d & e represents a biological repeat. Means ± SDs are shown. P values shown are calculated using one-way Anova. Source data for panels b–e are provided as Source Data files.

Fig. 4. Peripheral blood in G6 mice with mTert and hmTert alleles.

a Whole blood cell counts in adult mice of 3–6 months by hematology analyses. b Lymphocyte counts in peripheral blood. Cells were stained using antibodies and analyzed by flow cytometry. Each datapoint represents one animal. Means ± SDs are indicated. P values shown are calculated using one-way Anova. Source data are provided as Source Data files.

Fig. 5. Small intestines of G6 mice with mTert and hmTert alleles.

a Histopathology of small intestines of adult mice of 8–10 months. The bar indicates 100 µm. b The expression of genes regulating cellular senescence and proliferation in small intestine. Each column represents an individual mouse. Relative mRNA levels were determined by qRT-PCR and normalized to 18S rRNA. Means ± SDs are shown. Source data are provided as a Source Data file.

Fig. 6. Telomere length homeostasis in mice during Tert^h/- intercrosses.

a Breeding strategy. Tert^h/- progeny from G4 Tert^h/- parents were intercrossed. b Telomere length as determined by Flow-FISH. Splenocytes from 2-month-old mice were used for the analyses. P value within the groups of G4.14, G4.15, and G4.16 was calculated using one-way ANOVA. c Body weight of 8-week-old male and female mice. d Litter sizes. e Testis weight of mice at 10–15-week age. f Flow-FISH comparing telomere lengths of Tert^h/h, Tert^h/-, and Tert^-/- littermates. Each datapoint in panels b–f represents one animal. Means ± SDs are shown. n ≥ 3. g TRF analysis. Splenocyte genomic DNAs were digested with HinfI and RsaI, followed by 0.6% Agarose gel electrophoresis and Southern blotting. Sizes are indicated on the left (kb). MW, molecular weight marker. NHF (P11), passage 11 normal human foreskin fibroblasts. A longer exposure is shown in Supplementary Fig. 8a. h Genotype ratios of progeny in intercrosses at 7, 21, and 56 postnatal days. n = 21 ~ 93. Source data for panels b–h are provided as Source Data files.

Fig. 7. Telomere length homeostasis during incrosses of Tert^h/h mice.

a Breeding schemes. Tert^h/h progeny from G4, G4.8, and G4.14 Tert^h/- parents were successively incrossed. b Relative telomere signals. Telomere signals were determined by Flow-FISH and normalized to that of wildtype C57BL/6 J mice (50 kb). n ≥ 3. c TRF analysis. A longer exposure is shown in Supplementary Fig. 8b. d Body weight. e Litter sizes. f Testis weight. Each data point represents one animal. Means ± SDs are shown in panels b, & d–f. Source data for panels b–f are provided as Source Data files.

Fig. 8. Comparison of mTert and hmTert alleles.

a Breeding strategy. G6 Tert^+/- and Tert^h/- mice from Fig. 2a were independently intercrossed. b Relative telomere signals as determined by Flow-FISH and normalized to that of wildtype C57BL/6 J mice. c, Body weight. d Litter sizes. e Testis weight. Each data point represents one animal. Means ± SDs are shown. Source data for panels b–e are provided as Source Data files.

Fig. 9. Dextran sulfate sodium (DSS)-induced colitis in mice.

a Experimental strategy. 7–8-month-old Tert^+/+ (wildtype C57BL/6 J) and Tert^h/h (G4.8 h) mice were given drinking water with or without 3% DSS for 6 days, followed by 1 day of pure drinking water. Intraperitoneal EdU injection was performed 2 h before tissue collection. b Representative images of colons and spleens following DSS treatment. c Spleen weight. Spleen weight was normalized to the body weight of each mouse. Each data point represents one animal. n = 4. d EdU staining of colon crypt sections. Colon tissues were labeled with anti-EdU (white) and E-cadherin (green) antibodies, as well as Hoechst dye for nuclear staining (blue). Scale bar represents 100 µm. Representative images are shown. e Quantification of EdU-positive cells. Each datapoint in panel e represents the average number of EdU-positive cells per colon crypt in 30 crypts from one animal. n = 6. Means ± SDs are shown in panels c, e. P values, calculated using two-way Anova, are shown. Source data for panels c and e are provided as Source Data files.