Humanization of the mouse Tert gene reset telomeres to human length

Abstract

Telomeres undergo shortening with each cell division, serving as biomarkers of human aging, which is characterized by short telomeres and restricted telomerase expression in adult tissues. Contrarily, mice, featuring their longer telomeres and widespread telomerase activity, present limitations as models for understanding telomere-related human biology and diseases. To bridge this gap, we engineered a mouse strain with a humanized mTert gene, hmTert, wherein specific non-coding sequences were replaced with their human counterparts. The hmTert gene, encoding the wildtype mTert protein, was repressed in adult tissues beyond the gonads and thymus, closely resembling the regulatory pattern of the human TERT gene. Remarkably, the hmTert gene rescued telomere dysfunction in late generations of mTert-knockout mice. Through successive intercrosses of Tert^h/- mice, telomere length progressively declined, stabilizing below 10-kb. Tert^h/h mice achieved a human-like average telomere length of 10-12 kb, contrasting with the 50-kb length in wildtype C57BL/6J mice. Despite shortened telomeres, Tert^h/h mice maintained normal body weight and cell homeostasis in highly proliferative tissues. Notably, colonocyte proliferation decreased significantly in Tert^h/h mice during dextran sodium sulfate-induced ulcerative colitis-like pathology, suggesting limitations on cellular renewal due to short telomeres. Our findings underscore the genetic determination of telomere homeostasis in mice by the Tert gene. These mice, exhibiting humanized telomere homeostasis, serve as a valuable model for exploring fundamental questions related to human aging and cancer.

Extended Data Figure 1.

Developmental expression of the hmTert gene in mouse tissues. a, The expression of hmTert and mTert genes during post-natal development of Tert^h/+ mice. b, hmTert and mTert expression during T cell activation. CD4⁺ and CD8⁺ T cells isolated from spleens of Tert^h/+ mice were co-stimulated with CD3/CD28 antibodies and total RNAs were isolated. Tert mRNA data were determined by qRT-PCR assay, normalized to 18S rRNA, and compared to those in Tert^h/+ ESCs (1.0). c, T cell proliferation. Resting T cells were stimulated with CD3/CD28 antibodies for 48–96 h and incubated with 10μM EdU for 1 h. Percentages of labeled cells were determined by flow cytometry.

Extended Data Figure 2.

Telomere length and genotype ratios of G5 and G6 mice in Figure 2. a and b, Telomere length as determined by Flow-FISH. Telomere fluorescent signals of representative mice are shown in (a) and the data are summarized in (b). c, Genotype ratios of born offspring from G5 parents. Each data point represents one litter.

Extended Data Figure 3.

Hematopoietic cells in adult mice of 3–6 months. Mice were bred in Fig. 3a. a and b, Lymphocyte counts in spleen (a) and bone marrow (b). Cells were stained using antibodies and analyzed by flow cytometry. Each data point represent one animal. Means and SDs are shown. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way Anova.

Extended Data Figure 4.

Blood cell counts of Tert^h/h mice. a, Whole blood counts. b, White blood cell counts in peripheral blood. Cells were stained using antibodies and analyzed by flow cytometry. Each data point represents one animal. Means and SDs are shown.

Extended Data Figure 5.

Comparison of mTert and hmTert alleles. a, Breeding strategy. G6 Tert^+/− and Tert^h/− mice from Fig. 3a were independently intercrossed. b, Relative telomere signals as determined by Flow-FISH and normalized to that of wildtype C57BL/6J mice. c, Body weight. d, Litter sizes. e, Testis weight. Each data point represents one animal. Means and SDs are shown.

Figure 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. 0.5μg protein extracts from 4-month-old mice were used except for thymus (0.12μg). +, h, and − refer to mTert, hmTert, and mTert-KO alleles, respectively. 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. Mean and standard deviations (SDs) are shown. ***, p<0.001, two tailed student’s t test. 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).

Figure 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/6J 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), Tert^h/− and Tert^−/− (blue), Tert^−/− and Tert^−/− (black) 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 from 3–5 mice in each group. i and 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. Means and SDs are shown.

Figure 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/6J 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), Tert^h/− and Tert^−/− (blue), Tert^−/− and Tert^−/− (black) 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 from 3–5 mice in each group. i and 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. Means and SDs are shown.

Figure 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. f, Whole blood cell counts in adult mice of 3–6 months by hematology analyses. g, Lymphocyte counts in peripheral blood. Cells were stained using antibodies and analyzed by flow cytometry. Means and SDs are shown. ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way Anova. h, Histopathology of small intestines of adult mice of 8–10 months. The bar indicates 100 μm. i, 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 and SDs are shown.

Figure 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. f, Whole blood cell counts in adult mice of 3–6 months by hematology analyses. g, Lymphocyte counts in peripheral blood. Cells were stained using antibodies and analyzed by flow cytometry. Means and SDs are shown. ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way Anova. h, Histopathology of small intestines of adult mice of 8–10 months. The bar indicates 100 μm. i, 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 and SDs are shown.

Figure 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. f, Whole blood cell counts in adult mice of 3–6 months by hematology analyses. g, Lymphocyte counts in peripheral blood. Cells were stained using antibodies and analyzed by flow cytometry. Means and SDs are shown. ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way Anova. h, Histopathology of small intestines of adult mice of 8–10 months. The bar indicates 100 μm. i, 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 and SDs are shown.

Figure 4.

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. 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. g, TRF analysis. Splenocyte genomic DNAs were digested with HinfI and RsaI, followed by 0.6% Agraose gel electrophoresis and Southern blotting. Sizes are indicated on the left (kb). MW, molecular weight marker. NHF (P11), passage 11 normal human foreskin fibroblasts. h, Genotype ratios of progeny in intercrosses at 7, 21, and 56 postnatal days. Means and SDs are shown in panels B-F.

Figure 5.

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/6J mice (50 kb). c, TRF analysis. d, Body weight. e, Litter sizes. f, Testis weight. Each data point represents one animal. Means and SDs are shown.

Figure 5.

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/6J mice (50 kb). c, TRF analysis. d, Body weight. e, Litter sizes. f, Testis weight. Each data point represents one animal. Means and SDs are shown.

Figure 6.

Dextran sulfate sodium (DSS)-induced colitis in mice. a, Experimental strategy. 7–8-month-old Tert^+/+ (wildtype C57BL/6J) and Tert^h/h (G4.8h) 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 hours 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. 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). Images were captured using a Zeiss Image M2 microscope. Representative images are shown. e, Quantification of EdU-positive cells. Each data point represents the average number of EdU-positive cells per colon crypt in 30 crypts from one animal. ns, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001; N = 6; two-way Anova.