Telomerase reverse transcriptase gene knock-in unleashes enhanced longevity and accelerated damage repair in mice

Abstract

While previous research has demonstrated the therapeutic efficacy of telomerase reverse transcriptase (TERT) overexpression using adeno-associated virus and cytomegalovirus vectors to combat aging, the broader implications of TERT germline gene editing on the mammalian genome, proteomic composition, phenotypes, lifespan extension, and damage repair remain largely unexplored. In this study, we elucidate the functional properties of transgenic mice carrying the Tert transgene, guided by precise gene targeting into the Rosa26 locus via embryonic stem (ES) cells under the control of the elongation factor 1α (EF1α) promoter. The Tert knock-in (TertKI) mice harboring the EF1α-Tert gene displayed elevated telomerase activity, elongated telomeres, and extended lifespan, with no spontaneous genotoxicity or carcinogenicity. The TertKI mice showed also enhanced wound healing, characterized by significantly increased expression of Fgf7, Vegf, and collagen. Additionally, TertKI mice exhibited robust resistance to the progression of colitis induced by dextran sodium sulfate (DSS), accompanied by reduced expression of disease-deteriorating genes. These findings foreshadow the potential of TertKI as an extraordinary rejuvenation force, promising not only longevity but also rejuvenation in skin and intestinal aging.

Keywords: Tert knock‐in; damage repair; lifespan extension; telomerase reverse transcriptase; transgenic mice.

FIGURE 1

Generation, identification and phenotype of TertKI mice. (a) The schematic diagram illustrates the successful generation of TertKI mice that were stably inherited for four consecutive generations. The EF1α‐mTert transgene was targetedly integrated into the Rosa26 locus via ES cells, which involved construction of a targeting vector, ES cell culture, transfection, identification and proliferation of targeted ES cells. The targeted ES cells were then used to produce chimeric mice by injection into blastocysts. Subsequent breeding of the chimeric mice resulted in the birth of offspring carrying the modified gene. The removal of the Neo ^r gene 3′ to the mTert gene led to the production of F0 heterozygous and F1 homozygous mice lacking Neo ^r , while the deletion of the Stop sequence between the EF1α promoter and mTert cDNA resulted in G0 heterozygous and G1–G5 homozygous mice. (b) Genotype identification was conducted on the G5 (5th generation) TertKI mice using three sets of primers. Tert_F1 and 3′Arm_R primers amplify 520 bp from Neo‐Rox‐deleted allele but 4315 bp prior to deletion. 5′Arm_F3 and 3′Arm_R primers amplify 292 bp fragment from the WT Rosa26 locus only, whereas the length of DNA was 9965 bp in the original knock‐in construct and 6170 bp after the Neo‐Rox deletion. The primer pair of EF1α_F2 and Tert_R3 primers detect 472 bp from lox‐STOP‐removed allele but 1349 bp from original insert. Therefore, G5 homozygous EF1α‐mTert ^flox/flox mice (as shown in pups 1–6) were anticipated to display 520 bp (left) and 472 bp (right) PCR products, G0 heterozygous mice (EF1α‐mTert ^flox/+ ) should exhibit three bands of 520 bp, 292 bp (left) and 472 bp (right), whereas wild‐type (WT) mice were expected to be positive for 292 bp (left) DNA only. (c–e) A comparative analysis was performed to assess mTert gene expression, telomerase activity, and telomere length in TertKI mice and WT mice. The mRNA expression level of mTert gene (c), telomerase activity (d), and telomere length (e) in various organs and tissues of TertKI and WT mice aged 6 weeks were determined. Evaluations were conducted in six‐week‐old TertKI mice (in blue), and WT counterparts (in red), on a variety of organs and tissues (lung, kidney, liver, heart, muscle, spleen, brain and skin derived from three germ layers. Data are presented as mean ± standard deviation (SD). Statistical differences are represented on the graph, with p < 0.05 indicating a significant difference and p < 0.01 representing a highly significant difference.

FIGURE 2

Effects of Tert overexpression on lifespan and antioxidant capacity in mice. (a) Kaplan–Meier survival curve showing prolonged lifespan of G1–G5 TertKI mice compared to WT and EIIa‐Cre mice. (b–f) Individual survival curves for each generation of TertKI mice (G1–G5) compared to WT and EIIa‐Cre mice. (g) Kaplan–Meier survival curve separated by gender, showing comparable lifespan of female (n = 47) and male (n = 51) TertKI mice. Statistical differences are represented on the graph, with p < 0.05 indicating a significant difference. (h) The protein expression levels of Tert in the liver were evaluated through Western blot analysis (n = 4). (i) Hepatic GSH levels in WT (red) and TertKI (blue) mice (n = 3). (j) SOD activity in the liver of WT (red) and TertKI (blue) mice (n = 3). (k) Western blot analysis of p53 protein expression and quantification in liver of WT (red) and TertKI (blue) mice (n = 6). Data are presented as mean ± standard deviation (SD). Statistical differences are represented on the graph, with p < 0.05 indicating a significant difference and p < 0.01 representing a highly significant difference.

FIGURE 3

Effects of Tert overexpression on skin wound healing and the expression of inflammatory and growth factors. (a) The protein expression levels of Tert in the skin of both WT (C57BL/6) and TertKI (G3) mice (over 8 weeks old) were assessed using Western blot analysis. (b) The wound healing rate in acute skin wounds of WT and TertKI mice at 0, 2, 4, 5 and 7 days (n = 6). (c) Hydroxyproline levels surrounding the wounds in WT and TertKI mice (n = 5). (d) In vitro measurement of fibroblast wound healing ability from WT and TertKI mice (n = 6). Fibroblast cells were harvested from both WT and Tert‐overexpressing (TertKI) mice, then cultured under controlled conditions. An artificial wound was created within the fibroblast cell layer to simulate a real wound scenario. Subsequently, images were taken at designated time points (0 and 24 h) to monitor the response of fibroblast cells from both groups to the artificial wound. (e) The mRNA expression levels of inflammatory factors (Il1β and Tnfα) and growth factors (Fgf1, Col1α1 and Col3α1) around the wound of mouse skin were measured using RT‐qPCR. The expression of each gene in TertKI mice was calculated relative to its expression in WT mice on the same day. The gene expression in WT mice was normalized to 100% every day and is represented by the red dotted line in this panel. (f) The protein expression levels of Fgf7, Vegf, and Wnt‐1 were assessed using western blot from three independent experiments. The skin samples were collected from mice different from those used to measure wound healing. Thus, we ensured that the collected tissue samples did not interfere with the wound healing measurements, and we strictly distinguished the mice used for RNA and protein extraction from those used for wound healing assessments. Protein expression quantification of each band was calculated relative to the sample of WT mice on day 0. The protein expression in WT mice on day 0 was normalized to 100% and is shown as the red dotted line in this panel. Data are presented as mean ± standard deviation (SD). Statistical differences are represented on the graph, with p < 0.05 indicating a significant difference and p < 0.01 representing a highly significant difference.

FIGURE 4

Effects of Tert overexpression on DSS‐induced colitis. (a) The protein expression levels of Tert in the colon of both WT and TertKI mice were assessed using Western blot analysis. (b–e) The WT (C57BL/6) and TertKI (G5) mice (both over 8 weeks old) were subjected to treatment with either regular drinking water or water containing 3% DSS for a duration of 7 days to induce colitis. The mice were divided into four groups: WT‐Blank (n = 8), WT DSS‐induced (n = 8), TertKI‐Blank (n = 4), and TertKI DSS‐induced (n = 4). Various colitis‐related parameters were assessed and analyzed, including the colon length (b), loose stools score, bloody stool score (c), colon pathology (d), and western blot was conducted three times to assess the protein expression levels of Tgf‐β1, Wnt‐1 and β‐Catenin (e). Data are presented as mean ± standard deviation (SD). Statistical differences are represented on the graph, with p < 0.05 indicating a significant difference and p < 0.01 representing a highly significant difference.