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WO2024229085A2 - Modèle de souris avec télomère humanisé - Google Patents

Modèle de souris avec télomère humanisé Download PDF

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Publication number
WO2024229085A2
WO2024229085A2 PCT/US2024/027191 US2024027191W WO2024229085A2 WO 2024229085 A2 WO2024229085 A2 WO 2024229085A2 US 2024027191 W US2024027191 W US 2024027191W WO 2024229085 A2 WO2024229085 A2 WO 2024229085A2
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combination
disease
hmtert
transgenic mouse
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WO2024229085A3 (fr
Inventor
Jiyue ZHU
Shuwen Wang
Fan Zhang
De CHENG
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Washington State University WSU
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Washington State University WSU
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases

Definitions

  • telomeres play key roles in the development of many human diseases, especially age-related disorders and cancer. Generally, telomeres are replenished by telomerase, a ribonucleoprotein complex containing TERT, TERC, and accessory proteins. Humans are bom with 10-15 kb telomeres and adults have 5-15 kb of telomeres. Telomerase activity is very low or undetectable in most adult human tissues. As a result, telomeres are progressively shortened upon successive cell divisions and become exhausted in aged somatic tissues, triggering replicative senescence. Thus, telomeres can be observed to functioning as an aging clock in humans.
  • telomere mutations in human telomerase genes lead to dyskeratosis congenita, a prototypical telomere biology disorder that presents as a multi-system syndrome with a broad spectrum of clinical manifestations, including aplastic anemia and cancer.
  • Short telomere-induced replicative senescence in human cells also functions as a tumor- suppressing mechanism.
  • telomere regulation and telomere length Important differences exist in telomerase regulation and telomere length among different mammalian species. In humans, telomerase expression is restricted to a small number of organs (e.g., testis, ovary, and thymus) and telomeres are exhausted in many aged tissues. In contrast, a comparative study of over 60 mammals showed that many mammals, including mice, do not have telomere-mediated replicative aging.
  • Telomerase expression in mice is less restricted, with most tissues expressing significant levels of Tert mRNA and telomerase activity.
  • Laboratory inbred strains such as C57BL/6, have long telomeres.
  • Telomerase-null mice Tet or Terc-KO
  • mice can survive up to six generations with no discernible phenotypes in early generations, indicating that mice have adequate telomere reserves that are not exhausted for multiple generations without telomerase.
  • Mouse models of human diseases have become a central part of biomedical research. Laboratory mice provide the most experimentally accessible mammalian models that share genes, organs, and systemic physiology with humans.
  • mouse models fail to mimic human disease progression, posing translational challenges and limiting their use for human disease research. This facet may contribute to the high failure rates of human clinical trials, particularly in oncology, predicating the need for improved preclinical data from animal models. Therefore, there exists a need for new mouse models for evaluating human diseases and treatments thereof. In particular, mouse models that can be utilized for testing treatments of age-related disorders and cancers are very desirable.
  • the present disclosure provides a transgenic mouse and associated methods thereof for use as a mouse model.
  • the transgenic mouse described herein comprises a humanized mouse telomerase htnTerf) gene contained in a background murine germline that provides shortened telomeres that can replicate humans and various human disease states.
  • the transgenic mice described herein comprise a humanized mTert allele (JimTert) that can provide human-like telomere homeostasis.
  • JimTert humanized mTert allele
  • Tert mRNA expression and telomerase activity in these mice were distinct from those in wildtype mice but remarkably similar to those in humans.
  • the hmTert gene was able to rescue telomere deficiency when it was crossed into the fifth generation of mTert knockout (KO) mice.
  • KO mTert knockout mice.
  • the introduction of hmTert allele led to a recalibration of telomere equilibrium length in these mice, providing shorter telomere length compared to wildtype mice but resembling human telomere lengths.
  • FIGURES A- ID show the hmTert gene and its expression in mice.
  • FIGURE 1A shows the 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.
  • FIGURE IB shows telomerase expression in tissues from Tert +I- and Tert hl- littermates. Telomerase activities were determined by TRAP assay.
  • FIGURE 1C shows 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.
  • FIGURE ID shows hTERT mRNA expression in human tissues.
  • FIGURES 2A-2C show the developmental expression of the hmTert gene in mouse tissues. Where FIGURE 2A shows the expression of hmTert and mTert genes during post-natal development of Tert Mi mice.
  • FIGURE 2B shows the hmTert and mTert expression during T cell activation.
  • CD4 + and CD8 + T cells isolated from spleens of Tert h ' mice were costimulated with CD3/CD28 antibodies and total RNAs were isolated.
  • FIGURE 2C shows T cell proliferation. Resting T cells were stimulated with CD3/CD28 antibodies for 48-96 h and incubated with lOpM EdU for 1 h. Percentages of labeled cells were determined by flow cytometry.
  • FIGURES 3A-3J show the functions of the hmTert gene in mice.
  • FIGURE 3A shows the breeding strategy. Telomere length of splenocytes from 2-month-old Terl +I ⁇ , Terl hl ⁇ , and Tert -1- mice were determined by Flow-FISH, as shown in FIGURE 3B and in FIGURE 3C, the telomere restriction fragment (TRF) analysis is shown. Where FIGURE 3B shows telomere Flow-FISH. Telomere signals were detected by hybridization to FAM-
  • FIGURE 3C shows TRF analysis. Splenocyte genomic DNAs were digested with Hinfl and Rsal, followed by pulsed-field gel electrophoresis and Southern blotting. Positions of size markers are shown on the left (kb).
  • FIGURE 3D shows the litter sizes of breeding between Tert +I ⁇ and Tert -1- (red), Tert hl ⁇ and Terr 1- (blue), Terr 1- and Terr 1- (black) mice.
  • FIGURE 3E shows the body weight of male (upper) and female (lower) mice at 8-week of age.
  • FIGURE 3F shows the testis weight of mice at 10-15-week age.
  • FIGURE 3G shows the H&E staining of seminiferous tubules in testes from Tert +I ⁇ , Tert 1 ' 1- , and Tert -1- mice. Yellow arrowheads indicate aberrant tubules.
  • FIGURE 3H shows the average percentages of aberrant seminiferous tubules in testes from 3-5 mice in each group.
  • FIGURE 31 and FIGURE 3J shows the 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, shown in FIGURE 21 and G5, the mice are shown in FIGURE 3J. P- values of survival curve comparisons were calculated using logrank test. Means and SDs are shown.
  • FIGURES 4A-4E shows a comparison of G6 mice with mTert and hmTert alleles.
  • FIGURE 4A shows the mouse breeding scheme.
  • FIGURE 4B shows TRF analysis of representative animals. Splenocyte genomic DNAs were digested with Hinfl and Rsal, followed by pulsed-field gel electrophoresis and Southern blotting.
  • FIGURE 4C shows Kaplan-Meier survival curves of the mice. P- values comparing indicated paired curves were determined using logrank tests.
  • FIGURE 4D shows the body weight of male (upper) and female (lower) mice at 8-week of age.
  • FIGURE 4E shows the testis weight of mice at 10-15-week age.
  • FIGURE 4F shows whole blood cell counts in adult mice of 3-6 months by hematology analyses.
  • FIGURE 4G shows 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.
  • FIGURE 4H shows the histopathology of small intestines of adult mice of 8-10 months. The bar indicates 100 pm.
  • FIGURE 41 shows 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.
  • FIGURES 5A-5C show telomere length and genotype ratios of G5 and G6 mice in from FIGURE 4.
  • FIGURE 5A and FIGURE 5B show telomere length as determined by Flow-FISH. Telomere fluorescent signals of representative mice are shown in FIGURE 5A and the data are summarized in FIGURE 5B.
  • FIGURE 5C shows the genotype ratios of born offspring from G5 parents. Each data point represents one litter.
  • FIGURES 6A-6B show hematopoietic cells in adult mice of 3-6 months. Mice were bred FIGURE 4.
  • FIGURE 6 A shows lymphocyte counts in spleen and FIGURE 6B, the lymphocyte counts in bone marrow. 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.
  • FIGURES 7A-7H show telomere length homeostasis in mice during Tert M intercrosses. FIGURE 7A shows the breeding strategy.
  • FIGURE 7B shows telomere length as determined by Flow-FISH. Splenocytes from 2-month-old mice were used for the analyses.
  • FIGURE 7C shows the body weight of 8-week-old male and female mice.
  • FIGURE 7D shows litter sizes.
  • FIGURE 7E shows the testis weight of mice at 10- 15- week age.
  • FIGURE 7F shows the Flow- FISH comparing telomere lengths of Tert h/h , Tert h ' ⁇ , and Tert ⁇ ' ⁇ littermates.
  • FIGURE 7G shows the TRF analysis.
  • FIGURE 7H shows the genotype ratios of progeny in intercrosses at 7, 21, and 56 postnatal days. Means and SDs are shown in FIGURES 7B-7F.
  • FIGURES 8A-8F show telomere length homeostasis during incrosses of Tert h/h mice.
  • FIGURE 8A shows the breeding schemes. Tert Wh progeny from G4, G4.8, and G4.14 Tert h ' parents were successively incrossed.
  • FIGURE 8B shows the relative telomere signals. Telomere signals were determined by Flow-FISH and normalized to that of wildtype C57BL/6I mice (50 kb).
  • FIGURE 8C shows TRF analysis.
  • FIGURE 8D shows body weight.
  • FIGURE 8E shows litter sizes.
  • FIGURE 8F shows testis weight. Each data point represents one animal. Means and SDs are shown.
  • FIGURES 9A-9B show blood cell counts of Tert Ml mice.
  • FIGURE 9A shows whole blood counts.
  • FIGURE 9B shows 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.
  • FIGURES 10A-10E show a comparison of mTert and hmTert alleles.
  • FIGURE 10A shows the breeding strategy. G6 Tert +I ⁇ and Tert h ' ⁇ mice from FIGURE 4 A were independently intercrossed.
  • FIGURE 10B shows the relative telomere signals as determined by Flow-FISH and normalized to that of wildtype C57BL/6J mice.
  • FIGURE 10C shows body weight.
  • FIGURE 10D shows the litter sizes.
  • FIGURE 10E shows the testis weight. Each data point represents one animal. Means and SDs are shown.
  • FIGURES 11A-11E show dextran sulfate sodium (DSS)-induced colitis in mice.
  • FIGURE 11 A shows the general 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.
  • FIGURE 1 IB shows representative images of colons and spleens following DSS treatment.
  • FIGURE 11C shows spleen weight. Spleen weight was normalized to the body weight of each mouse.
  • FIGURE 1 ID shows EdU staining of colon crypt sections.
  • FIGURE 12 shows the sequence of SEQ ID NO: 1.
  • FIGURE 12 shows the sequence of SEQ ID NO:2, which is the sequence of the hmTert knock-in locus.
  • the upper case represents mouse sequence while the bold lower case represents human sequence.
  • the 5’IR (5’ intergenic region) is distinctly delineated with an underline, the In2 (intron 2) is highlighted with a double underline, and the In6 (intron 6) is emphasized with a wave underline.
  • FIGURE 14 shows the sequence of SEQ ID NO:3, which displays hmTert with introns 2 and 6.
  • a transgenic mouse comprises i) a background murine germline and ii) a humanized mouse telomerase (hmTert gene contained in the background murine germline.
  • the transgenic mouse is a humanized transgenic mouse. In an embodiment, the transgenic mouse is a humanized transgenic mouse. In an embodiment, the hmTert gene comprises SEQ ID NO:1. In an embodiment, the hmTert gene consists essentially of SEQ ID NO:1. In an embodiment, the hmTert gene consists of SEQ ID NO:1.
  • the hmTert gene has at least 80% sequence identity to SEQ ID NO: 1. In an embodiment, the hmTert gene has at least 85% sequence identity to SEQ ID NO:1. In an embodiment, the hmTert gene has at least 90% sequence identity to SEQ ID NO: 1. In an embodiment, the hmTert gene has at least 95% sequence identity to SEQ ID NO:1. In an embodiment, the hmTert gene has at least 96% sequence identity to SEQ ID NO:1. In an embodiment, the hmTert gene has at least 97% sequence identity to SEQ ID NO:1. In an embodiment, the hmTert gene has at least 98% sequence identity to SEQ ID NO:1. In an embodiment, the hmTert gene has at least 99% sequence identity to SEQ ID NO:1.
  • the hmTert gene comprises SEQ ID NO:1, and SEQ ID NO:1 comprises intron 2 of the human TERT gene. In an embodiment, the hmTert gene comprises SEQ ID NO: 1, and SEQ ID NO:1 comprises intron 6 of the human TERT gene. In an embodiment, the hmTert gene comprises SEQ ID NO:1, and SEQ ID NO:1 comprises intron 2 and intron 6 from the human TERT gene.
  • the hmTert gene comprises SEQ ID NO:1, and SEQ ID NO:1 comprises a 5’ intergenic region (5 ’IR) of the human TERT gene.
  • the hmTert gene comprises SEQ ID NO:1, and SEQ ID NO:1 comprises a 5’IR and intron 2 of the human TERT gene.
  • the hmTert gene comprises SEQ ID NO: 1, and SEQ ID NO:1 comprises a 5’IR and intron 6 of the human TERT gene.
  • the hmTert gene comprises SEQ ID NO: 1, and SEQ ID NO: 1 comprises a 5’IR and intron 2 and intron 6 of the human TERT gene.
  • the hmTert gene comprises SEQ ID NO:2. In an embodiment, the hmTert gene consists essentially of SEQ ID NO:2. In an embodiment, the hmTert gene consists of SEQ ID NO:2.
  • the hmTert gene has at least 80% sequence identity to SEQ ID NO:2. In an embodiment, the hmTert gene has at least 85% sequence identity to SEQ ID NO:2. In an embodiment, the hmTert gene has at least 90% sequence identity to SEQ ID NO:2. In an embodiment, the hmTert gene has at least 95% sequence identity to SEQ ID NO:2. In an embodiment, the hmTert gene has at least 96% sequence identity to SEQ ID NO:2. In an embodiment, the hmTert gene has at least 97% sequence identity to SEQ ID NO:2. In an embodiment, the hmTert gene has at least 98% sequence identity to SEQ ID NO:2. In an embodiment, the hmTert gene has at least 99% sequence identity to SEQ ID NO:2.
  • the hmTert gene comprises SEQ ID NO:2, and SEQ ID NO:2 comprises intron 2 of the human TERT gene. In an embodiment, the hmTert gene comprises SEQ ID NO:2, and SEQ ID NO:2 comprises intron 6 of the human TERT gene. In an embodiment, the hmTert gene comprises SEQ ID NO:2, and SEQ ID NO:2 comprises intron 2 and intron 6 from the human TERT gene.
  • the hmTert gene comprises SEQ ID NO:2, and SEQ ID NO:2 comprises a 5’ intergenic region (5’IR) of the human TERT gene.
  • the hmTert gene comprises SEQ ID NO:2, and SEQ ID NO:2 comprises a 5’IR and intron 2 of the human TERT gene.
  • the hmTert gene comprises SEQ ID NO: 2, and SEQ ID N0:2 comprises a 5’IR and intron 6 of the human TERT gene.
  • the hmTert gene comprises SEQ ID NO:2, and SEQ ID NO:2 comprises a 5’IR and intron 2 and intron 6 of the human TERT gene.
  • the hmTert gene comprises SEQ ID NO:3. In an embodiment, the hmTert gene consists essentially of SEQ ID NO:3. In an embodiment, the hmTert gene consists of SEQ ID NO:3.
  • the hmTert gene has at least 80% sequence identity to SEQ ID NO:3. In an embodiment, the hmTert gene has at least 85% sequence identity to SEQ ID NO:3. In an embodiment, the hmTert gene has at least 90% sequence identity to SEQ ID NO:3. In an embodiment, the hmTert gene has at least 95% sequence identity to SEQ ID NO:3. In an embodiment, the hmTert gene has at least 96% sequence identity to SEQ ID NO:3. In an embodiment, the hmTert gene has at least 97% sequence identity to SEQ ID NO:3. In an embodiment, the hmTert gene has at least 98% sequence identity to SEQ ID NO:3. In an embodiment, the hmTert gene has at least 99% sequence identity to SEQ ID NO:3.
  • the hmTert gene comprises SEQ ID NO:3, and SEQ ID NO:3 comprises intron 2 of the human TERT gene. In an embodiment, the hmTert gene comprises SEQ ID NO:3, and SEQ ID NO:3 comprises intron 6 of the human TERT gene. In an embodiment, the hmTert gene comprises SEQ ID NO:3, and SEQ ID NO:3 comprises intron 2 and intron 6 of the human TERT gene.
  • the hmTert gene comprises SEQ ID NO:3, and SEQ ID NO:3 comprises a 5’ intergenic region (5’IR) of the human TERT gene.
  • the hmTert gene comprises SEQ ID NO:3, and SEQ ID NO:3 comprises a 5’IR and intron 2 of the human TERT gene.
  • the hmTert gene comprises SEQ ID NO:3, and SEQ ID NO:3 comprises a 5’IR and intron 6.
  • the hmTert gene comprises SEQ ID NO:3, and SEQ ID NO:3 comprises a 5’IR and intron 2 and intron 6 of the human TERT gene.
  • the background murine germline is from a species of Mus genus. In an embodiment, the background murine germline is a strain of a Mus muse ulus line. [0039] In an embodiment, the background murine germline is a substrain of C57BL/6 line. In an embodiment, the background murine germline is a substrain of J:NU. In an embodiment, the background murine germline is a substrain of NU/J. In an embodiment, the background murine germline is a substrain of 129Sl/SvImJ. In an embodiment, the background murine germline is a substrain of 129Xl/SvJ.
  • the background murine germline is a substrain of A/J. In an embodiment, the background murine germline is a substrain of AKR/J. In an embodiment, the background murine germline is a substrain of BALB/cByJ. In an embodiment, the background murine germline is a substrain of B ALB/cJ. In an embodiment, the background murine germline is a substrain of C3H/HeJ. In an embodiment, the background murine germline is a substrain of C57BL/6J. In an embodiment, the background murine germline is a substrain of C57BL/6NJ. In an embodiment, the background murine germline is a substrain of C57BL/10J.
  • the background murine germline is a substrain of CBA/J. In an embodiment, the background murine germline is a substrain of CBA/CaJ. In an embodiment, the background murine germline is a substrain of DBA/1J. In an embodiment, the background murine germline is a substrain of DBA/2J. In an embodiment, the background murine germline is a substrain of FVB/NJ. In an embodiment, the background murine germline is a substrain of C57BL/6J-4/? ⁇ - w, '7J.
  • the transgenic mouse comprises expression maintenance of the hmTert gene.
  • expression maintenance refers to the ability of a mouse to continue expressing a particular gene upon one or more subsequent generations of breeding.
  • the transgenic mouse comprises humanized telomere homeostasis.
  • humanized telomere homeostasis can refer to mice possessing telomerase expression that is suppressed in adult tissues (see, e.g., Figure 1) and/or that the telomeres comprise an average of telomere length from five to twenty (5-20) kilobase (kb).
  • the telomere homeostasis is maintained upon interbreeding for one or more generations.
  • the mouse comprises one or more telomeres, and the telomeres comprise an average telomere length from five to twenty (5-20) kilobase (kb). In an embodiment, the telomeres comprise an average telomere length from 5-20 kb. In an embodiment, the telomeres comprise an average telomere length from 5-15 kb. In an embodiment, the telomeres comprise an average telomere length from 5-10 kb. In an embodiment, the telomeres comprise an average telomere length from 6-20 kb. In an embodiment, the telomeres comprise an average telomere length from 6-18 kb.
  • kb kilobase
  • the telomeres comprise an average telomere length from 6-16 kb. In an embodiment, the telomeres comprise an average telomere length from 6-14 kb. In an embodiment, the telomeres comprise an average telomere length from 6-12 kb. In an embodiment, the telomeres comprise an average telomere length from 6-10 kb. In an embodiment, the telomeres comprise an average telomere length from 6-8 kb. In an embodiment, the telomeres comprise an average telomere length from 8-20 kb. In an embodiment, the telomeres comprise an average telomere length from 8-18 kb.
  • the telomeres comprise an average telomere length from 8-16 kb. In an embodiment, the telomeres comprise an average telomere length from 8-14 kb. In an embodiment, the telomeres comprise an average telomere length from 8-12 kb. In an embodiment, the telomeres comprise an average telomere length from 8-10 kb. In an embodiment, the telomeres comprise an average telomere length from 10-20 kb. In an embodiment, the telomeres comprise an average telomere length from 10-18 kb. In an embodiment, the telomeres comprise an average telomere length from 10-16 kb.
  • the telomeres comprise an average telomere length from 10-14 kb. In an embodiment, the telomeres comprise an average telomere length from 10-12 kb. In an embodiment, the telomeres comprise an average telomere length from 12-20 kb. In an embodiment, the telomeres comprise an average telomere length from 12-18 kb. In an embodiment, the telomeres comprise an average telomere length from 12-16 kb. In an embodiment, the telomeres comprise an average telomere length from 12-14 kb. In an embodiment, the telomeres comprise an average telomere length from 14-20 kb.
  • the telomeres comprise an average telomere length from 14-18 kb. In an embodiment, the telomeres comprise an average telomere length from 14-16 kb. In an embodiment, the telomeres comprise an average telomere length from 16-20 kb. In an embodiment, the telomeres comprise an average telomere length from 16-18 kb.
  • the telomeres comprise an average telomere length less than 20 kb. In an embodiment, the telomeres comprise an average telomere length less than 15 kb. In an embodiment, the telomeres comprise an average telomere length less than 10 kb.
  • a method for evaluating a treatment comprises the steps of i) administering the treatment to a transgenic mouse and ii) analyzing a response to the treatment in the transgenic mouse, wherein the response provides evaluation of the treatment.
  • the treatment is a drug.
  • the drug is used to treat aplastic anemia.
  • the drug is used to treat idiopathic aplastic anemia.
  • the drug is used to treat pulmonary fibrosis.
  • the drug is used to treat familial idiopathic pulmonary fibrosis.
  • the drug is used to treat dyskeratosis cogenita.
  • the drug is used to treat hepatic disease.
  • the drug is used to treat cirrhosis with inflammation.
  • the drug is used to treat nodular regenerative hyperplasia.
  • the drug is used to treat cancer.
  • the cancer is carcinoma with nonreciprocal translocations.
  • the cancer is colorectal cancer.
  • the cancer is esophageal cancer.
  • the cancer is head and neck squamous cell carcinoma.
  • the cancer is skin cancer.
  • the cancer is anorectal cancer.
  • the cancer is acute myeloid leukemia.
  • the cancer is leukemic transformation in myelodysplasia.
  • the cancer is secondary myelodysplasia.
  • the cancer is secondary leukemia.
  • the cancer is basal cell cancer of the skin.
  • the cancer is cancer of the lung. In an embodiment, the cancer is cancer of the bladder. In an embodiment, the cancer is cancer of the prostate. In an embodiment, the cancer is cancer of the cervix. In an embodiment, the cancer is glioblastoma. In an embodiment, the cancer is renal cell carcinoma. In an embodiment, the cancer is Barrett’s esophagus hepatocellular carcinoma.
  • the drug is used to treat inflammatory bowel disease.
  • the drug is used to treat chronic hepatitis B infection.
  • the drug is used to treat ulcerative colitis.
  • the drug is used to treat chronic graft- versus-host disease.
  • the drug is used to treat cardiovascular disease.
  • the treatment is radiation. In an embodiment, the treatment is surgery. In an embodiment, the treatment is a transplant. In an embodiment, the treatment is immunotherapy. In an embodiment, the treatment is gene therapy. In an embodiment, the treatment is biological engineering.
  • the treatment is a disease-causing agent.
  • the disease-causing agent is a pathogen.
  • the disease-causing agent is a carcinogen.
  • the disease-causing agent is a cancer cell.
  • the administering comprises an injection. In an embodiment, the administering comprises oral administration. In an embodiment, the administering comprises topical application. In an embodiment, the administering comprises inhalation. In an embodiment, the administering comprises radiation. In an embodiment, the administering comprises transplantation.
  • the response is an immune system response.
  • the response is tumorigenesis.
  • the response is senescence.
  • the response is cellular aging.
  • the response is a compromised innate immune response.
  • the response is a compromised adaptive immune response.
  • the response is apoptosis.
  • the response is oncogenic transformation.
  • the response is side effects.
  • the response is presence of disease.
  • the response is telomere length.
  • the response is telomerase activity.
  • the response is cellular metabolic activity.
  • the response is gene expression.
  • the response is RNA expression.
  • the response is protein expression.
  • the response is to cancer.
  • the cancer is melanoma.
  • the cancer is adrenal gland cancer.
  • the cancer is bladder cancer.
  • the cancer is bone cancer.
  • the cancer is blood cancer.
  • the cancer is brain cancer.
  • the cancer is breast cancer.
  • the cancer is cervical cancer.
  • the cancer is colon cancer.
  • the cancer is endometrium cancer.
  • the cancer is esophageal cancer.
  • the cancer is eye cancer.
  • the cancer is head and neck cancer.
  • the cancer is intestinal cancer.
  • the cancer is kidney cancer.
  • the cancer is liver cancer.
  • the cancer is lung cancer. In an embodiment, the cancer is mouth cancer. In an embodiment, the cancer is muscle cancer. In an embodiment, the cancer is nose cancer. In an embodiment, the cancer is ovarian cancer. In an embodiment, the cancer is pancreatic cancer. In an embodiment, the cancer is pituitary cancer. In an embodiment, the cancer is prostate cancer. In an embodiment, the cancer is skin cancer. In an embodiment, the cancer is stomach cancer. In an embodiment, the cancer is thymus cancer. In an embodiment, the cancer is thyroid cancer. In an embodiment, the cancer is testicular cancer. In an embodiment, the cancer is an immune system cancer. In an embodiment, the cancer is a nervous system cancer. In an embodiment, the cancer is a neuroendocrine system cancer.
  • the cancer is lymphoproliferative disease. In an embodiment, the cancer is a hematologic malignancy. In an embodiment, the cancer is a myelodysplastic syndrome. In an embodiment, the cancer is acute myeloid leukemia.
  • the response is to a rejuvenation or lifespan extension.
  • the rejuvenation or lifespan extension is a telomerase-mediated rejuvenation.
  • the rejuvenation or lifespan extension is epigenetic reprogramming.
  • the rejuvenation or lifespan extension is to a senolytic therapy.
  • the rejuvenation or lifespan extension is to an immunotherapy.
  • the response is skin appearance. In an embodiment, the response is hair loss/growth. In an embodiment, the response is nail aging/rejuvenation. In an embodiment, the response is to skincare products. In an embodiment, the response is to cosmetics. In an embodiment, the response is to hair graying. In an embodiment, the response is to alopecia. In an embodiment, the response is to hair loss treatment. In an embodiment, the response is to hyperpigmentation. In an embodiment, the response is to hypopigmentation. In an embodiment, the response is to abnormal skin pigmentation. In an embodiment, the response is to nail dystrophy. In an embodiment, the response is to adermatoglyphia.
  • the response is to shortened telomeres.
  • the shortened telomeres result from Coats Plus syndrome.
  • the shortened telomeres result from Fanconi anemia.
  • the shortened telomeres result from Revesz syndrome.
  • the shortened telomeres result from Hoyeraal- Hreidarsson syndrome.
  • the response is to telomere extension.
  • the telomere extension results from telomerase activation.
  • the telomere extension results from epigenetic reprogramming.
  • the response is to stem cell proliferation and maintenance.
  • the stem cell proliferation and maintenance comprises telomere-mediated stem cell failures manifested in lesions of skin, hematopoietic, gastrointestinal, and reproductive systems.
  • the response is to an immunologic system reaction. In an embodiment, the response is to a pharmaceutical event. In an embodiment, the response is to an environmental exposure. In an embodiment, the response is to a human lifestyle event.
  • the response is to a disease.
  • the disease is an aging or age-related disease.
  • the aging or age-related disease is senescence/aging.
  • the aging or age-related disease is longevity.
  • the aging or age-related disease is Dyskeratosis congenita (DC).
  • the aging or age-related disease is Werner syndrome.
  • the aging or age-related disease is Bloom syndrome.
  • the aging or age-related disease is Hutchinson-Gilford progeria syndrome.
  • the disease is a digestive system disease.
  • the digestive system disease is oral leukoplakia.
  • the digestive system disease is oesophageal structures and webs.
  • the digestive system disease is telangiectasias prone to hemorrhage.
  • the digestive system disease is villus atrophy.
  • the digestive system disease is enteropathy.
  • the disease is a lung disease.
  • the lung disease is pulmonary fibrosis (PF).
  • the lung disease is chronic obstructive pulmonary disease (COPD).
  • the lung disease is emphysema.
  • the lung disease is non-cystic fibrosis bronchiectasis.
  • the disease is a neurodegenerative disease.
  • the neurodegenerative disease is dementia.
  • the neurodegenerative disease is Alzheimer’ s disease (AD).
  • the neurodegenerative disease is Parkinson’s disease (PD).
  • the neurodegenerative disease is Huntington’s disease (HD).
  • the disease is an eye disease.
  • the eye disease is age-related macular degeneration.
  • the disease is a bone marrow disease.
  • the bone marrow disease is aplastic anemia.
  • the bone marrow disease is bone marrow failure.
  • the bone marrow disease is myelodysplastic syndrome
  • the disease is an immune system reaction.
  • the immune system reaction is an innate immune reaction.
  • the immune system reaction is an adaptive immune reaction.
  • the immune system reaction is immune cell senescence and exhaustion.
  • the immune system reaction is a B cell lymphopenia/immunodeficiency.
  • the immune system reaction is a T cell lymphopenia/immunodeficiency.
  • the immune system reaction is a natural killer (NK) cell lymphopenia/immunodeficiency.
  • the disease is an infectious disease.
  • the infectious disease is caused by HIV.
  • the infectious disease is caused by SARS-CoV-2.
  • the infectious disease is caused by a hepatitis virus.
  • the disease is an inflammatory disease.
  • the inflammatory disease is inflammatory bowel disease (IBD).
  • the inflammatory disease is Crohn’s disease (CD).
  • the inflammatory disease is ulcerative colitis (UC).
  • the inflammatory disease is rheumatoid arthritis.
  • the inflammatory disease is atherosclerosis.
  • the inflammatory disease is a chronic renal disease.
  • the disease is a metabolic disease.
  • the metabolic disease is chronic inflammation.
  • the metabolic disease is obesity.
  • the metabolic disease is dyslipidaemia.
  • the metabolic disease is hypertension.
  • the metabolic disease is insulin resistance.
  • the metabolic disease is diabetes.
  • the disease is a hepatic disease.
  • the hepatic disease is cirrhosis.
  • the hepatic disease is non-alcoholic fatty liver disease (NAFLD).
  • the hepatic disease is non-alcoholic steatohepatitis (NASH).
  • the hepatic disease is fibrosis.
  • the hepatic disease is primary biliary cirrhosis.
  • the hepatic disease is alcoholic liver disease.
  • the hepatic disease is nodular regenerative hyperplasia hepatopulmonary syndrome.
  • the hepatic disease is chronic viral hepatitis.
  • the disease is a cardiovascular disease.
  • the cardiovascular disease is cardiomyocyte hypertrophy.
  • the cardiovascular disease is fibrosis.
  • the cardiovascular disease is cardiac ischaemiareperfusion injury (1R1).
  • the cardiovascular disease is hypertrophic or dilated cardiomyopathy.
  • the cardiovascular disease is myocardial infarction.
  • the cardiovascular disease is atherosclerosis.
  • the disease is a musculoskeletal disease.
  • the musculoskeletal disease is osteopenia.
  • the musculoskeletal disease is osteoarthritis.
  • the musculoskeletal disease is rheumatoid arthritis.
  • the musculoskeletal disease is osteoporosis.
  • the musculoskeletal disease is skeletal fragility.
  • the musculoskeletal disease is avascular necrosis.
  • the musculoskeletal disease is Duchenne muscular dystrophy (DMD).
  • the disease is a kidney disease.
  • the kidney disease is an acute kidney injury.
  • the kidney disease is glomerulonephritis.
  • the kidney disease is diabetic nephropathy.
  • the kidney disease is polycystic kidney disease.
  • the kidney disease is kidney fibrosis.
  • the kidney disease is chronic kidney disease (CKD).
  • the disease is a reproductive disease.
  • the reproductive disease comprises testicular function.
  • the reproductive disease comprises ovarian function.
  • the reproductive disease comprises male infertility.
  • the reproductive disease comprises female infertility.
  • the response is a susceptibility and resiliency of human health in response to a pharmaceutical intervention.
  • the pharmaceutical intervention is rapamycin.
  • the pharmaceutical intervention is 17(3- oestradial.
  • the pharmaceutical intervention is senolytic strategies.
  • the pharmaceutical intervention is drug efficacy.
  • the pharmaceutical intervention is drug toxicity.
  • the pharmaceutical intervention is a drug-drug interaction.
  • the pharmaceutical intervention is a pharmacodynamic study.
  • the response is susceptibility and resiliency of human health in response to an environmental exposure.
  • the environmental exposure is air pollution.
  • the environmental exposure is food poisoning.
  • the environmental exposure is heavy metal.
  • the environmental exposure is hazardous working condition.
  • the environmental exposure is algae.
  • the environmental exposure is water pollution.
  • the environmental exposure is a mutagen.
  • the environmental exposure is radiation.
  • the environmental exposure is space travel.
  • the response is susceptibility and resiliency of human health in response to a lifestyle change.
  • the lifestyle change is dietary restriction.
  • the lifestyle change is cigarette smoking.
  • the lifestyle change is exercise.
  • the lifestyle change is physical work.
  • the lifestyle change is sleep deprivation.
  • the lifestyle change is night shift working condition.
  • the lifestyle change is chronic fatigue.
  • the lifestyle change is life stress.
  • the lifestyle change is psychological stress.
  • the lifestyle change is a psychological disorder.
  • the lifestyle change is mental health.
  • the lifestyle change is a psychiatric illness.
  • a method of generating a transgenic mouse comprises the steps of inserting an hmTert gene into a background murine germline to form a modified embryonic stem cell; and generating a first- generation mouse from the modified embryonic stem cell, wherein the first-generation mouse comprises the hmTert gene.
  • the method further comprises breeding one or more generations from the first-generation mouse to generate a progeny mouse.
  • the breeding one or more generations comprises controlled breeding with one or more of hmTert, mTert, and znTert-knockout strains.
  • the progeny mouse exhibits average telomere length of five to fifteen (5-15) kilobase.
  • a method of generating a transgenic mouse comprises a step of breeding a first mouse comprising an hmTert gene with a second mouse to generate a heterozygous mouse.
  • the method further comprises a step of breeding one or more generations from the heterozygous mouse.
  • a step of breeding one or more generations from the heterozygous mouse.
  • a transgenic mouse comprising i) a background murine germline and ii) a humanized mouse telomerase hmTert) gene contained in the background murine germline.
  • transgenic mouse of clause 1 any other suitable clause, or any combination of suitable clauses, wherein the transgenic mouse is a humanized transgenic mouse.
  • transgenic mouse of clause 1, any other suitable clause, or any combination of suitable clauses wherein the hmTert gene comprises SEQ ID NO:2, and wherein SEQ ID NO:2 comprises a 5’ intergenic region (5’IR).
  • transgenic mouse of clause 1, any other suitable clause, or any combination of suitable clauses wherein the hmTert gene comprises SEQ ID NO:3, and wherein SEQ ID NO:3 comprises intron 2 and intron 6.
  • transgenic mouse of clause 1, any other suitable clause, or any combination of suitable clauses wherein the hmTert gene comprises SEQ ID NO:3, and wherein SEQ ID NO:3 comprises a 5’IR and intron 2 and intron 6.
  • transgenic mouse of clause 1, any other suitable clause, or any combination of suitable clauses wherein the background murine germline is a strain of a Mus musculus line.
  • transgenic mouse of clause 1 any other suitable clause, or any combination of suitable clauses, wherein the background murine germline is a substrain of 129Sl/SvImJ.
  • transgenic mouse of clause 1, any other suitable clause, or any combination of suitable clauses wherein the background murine germline is a substrain of 129Xl/SvJ.
  • transgenic mouse of clause 1, any other suitable clause, or any combination of suitable clauses wherein the background murine germline is a substrain of C3H/HeJ.
  • transgenic mouse of clause 1, any other suitable clause, or any combination of suitable clauses wherein the mouse comprises one or more telomeres, and wherein the telomeres comprise an average telomere length from five to twenty (5-20) kilobase (kb).
  • telomeres comprise an average telomere length from 5-20 kb.
  • telomeres comprise an average telomere length from 5-15 kb.
  • telomeres comprise an average telomere length from 5-10 kb.
  • telomeres comprise an average telomere length from 6-20 kb.
  • telomeres comprise an average telomere length from 6-18 kb.
  • telomeres comprise an average telomere length from 6-16 kb.
  • telomeres comprise an average telomere length from 6-14 kb.
  • telomeres comprise an average telomere length from 6-12 kb.
  • telomeres comprise an average telomere length from 6-10 kb.
  • telomeres comprise an average telomere length from 6-8 kb.
  • telomeres comprise an average telomere length from 8-18 kb.
  • telomeres comprise an average telomere length from 8-16 kb.
  • telomeres comprise an average telomere length from 8-14 kb.
  • telomeres comprise an average telomere length from 8-12 kb.
  • telomeres comprise an average telomere length from 8-10 kb.
  • telomeres comprise an average telomere length from 10-20 kb.
  • telomeres comprise an average telomere length from 10-18 kb.
  • telomeres comprise an average telomere length from 10-16 kb.
  • telomeres comprise an average telomere length from 10-14 kb.
  • telomeres comprise an average telomere length from 10-12 kb.
  • telomeres comprise an average telomere length from 12-20 kb.
  • telomeres comprise an average telomere length from 12-18 kb.
  • telomeres comprise an average telomere length from 12-16 kb.
  • telomeres comprise an average telomere length from 12-14 kb.
  • telomeres comprise an average telomere length from 14-20 kb.
  • telomeres comprise an average telomere length from 14-16 kb.
  • telomeres comprise an average telomere length from 16-20 kb.
  • telomeres comprise an average telomere length from 16-18 kb.
  • telomeres comprise an average telomere length less than 20 kb.
  • telomeres comprise an average telomere length less than 15 kb.
  • telomeres comprise an average telomere length less than 10 kb.
  • a method for evaluating a treatment comprising steps of i) administering the treatment to a transgenic mouse and ii) analyzing a response to the treatment in the transgenic mouse, wherein the response provides evaluation of the treatment.
  • kidney disease chronic kidney disease
  • a method of generating a transgenic mouse comprising steps of:
  • a method of generating a transgenic mouse comprising a step of breeding a first mouse comprising an hmTert gene with a second mouse to generate a heterozygous mouse.
  • mRNA expression analyses were performed as previously described and data were normalized to 18S ribosomal RNA. Primer sequences are provided in Table 1, Table 2, and Table 3. Telomerase activities were determined using a modified telomeric repeat amplification protocol (TRAP) assay. Tissues and cell extracts were adjusted to same concentration and 0.5pg samples were used in each reaction. ESCs (Tert h/h , Tert + ' + , and Tert +/h ) served as positive controls.
  • TRIP modified telomeric repeat amplification protocol
  • Telomere lengths were measured using two independent methods. TRF analysis was described previously. Genomic DNAs were digested with Hinf I and Rsa I, and subjected to pulsed-field gel electrophoresis using CHEF-DR III Pulsed field Electrophoresis Systems (for telomeres over 20-kb) or regular 0.6% Agarose gel (for telomeres less than 20-kb), followed by Southern blotting using a (TTAGGGh-Biotin probe. Probe signal was developed with Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher). Telomere lengths were also measured by Flow-FISH. Telomere signals were detected by hybridization to FAM-(CCCTAA)3 oligonucleotide and converted to arbitrary units of molecular equivalents of soluble fluorescence.
  • CD4 + and CD8 + T cells were isolated from splenocytes using MojoSortTM T cells isolation kits (Biolegend, USA). Briefly, spleens were collected in RPMI-1640 medium (Gibco, USA) as soon as mice were sacrificed. The splenocytes were isolated through spleen crushing and filtration using a 70 pm strainer. Red blood cells were lysed with lx RBC lysis buffer, splenocytes were incubated with biotin- antibody cocktails on ice for 20 mins, and streptavidin nanobeads beads were added for 5 mins, followed by pulldown of non-T cells.
  • 6- well plates were coated by lOpg/ml anti-mouse CD3e antibody in PBS for 2 hours at 37°C.
  • the isolated CD4 or CD8 T cells were stimulated in CD3e antibody coated plates, together with added 2 pg/ml anti-mouse CD28 antibody for 24 h.
  • 10 ng/ml IL-2 was added at 48, 72 and 96 h and activated T cells were examined for cell proliferation using Click-iT® Plus EdU Flow Cytometry Assay Kit (ThermoFisher, USA) or harvested for gene expression analyses.
  • Fresh intestines were fixed in 10% formalin solution for 48 h, embedded in paraffin, sectioned at 5 pm, and stained with hematoxylin and eosin (H&E).
  • Mouse testes were fixed in Bouin’s fixative for 3 days and similarly processed. Slides were evaluated for aberrant seminiferous tubules in a blinded fashion and at least three mice of each experimental group were analyzed.
  • mice The whole blood samples were collected from submandibular veins of 3-6 months mice. Numbers of WBCs, RBCs, platelets, lymphocytes, neutrophils, and monocytes were determined on a Hemogram Analyzer (Abaxis HM5).
  • Peripheral blood, splenocytes, and femur bone marrow (BM) were collected from mice of 3-6 months.
  • Peripheral blood was washed twice with PBS by centrifugation in the presence of heparin, followed by removing RBCs with lx RBC lysis buffer (Thermo Fisher, USA). WBCs were counted using hemocytometers, stained with CD4, CD8, and CD19 antibodies, and analyzed by flow cytometry.
  • Splenocytes and bone marrow cells, flushed from femurs by syringes with 20 ml PBS buffer, were similarly processed for cell counting, FACS staining, and analyses.
  • Dextran sulfate sodium Cat. 160110, MP biomedical
  • Body weight of treated mice were monitored.
  • EdU labeling 150pl of 10 mg/ml EdU solution was injected intraperitoneally into mice two hours before sacrifice. Paraformaldehyde-fixed tissues were embedded in OCT matrix. EdU was detected using the Click-iT Plus EdU Cell Proliferation Kit (Alexa FluorTM 594, C 10639, ThermoFisher). Tissue sections were stained overnight with an anti-E-Cadherin antibody (1:250, Cat.
  • the hmTert gene containing the human 5’IR (23-kb), introns 2 (11-kb) and 6 (5.5-kb) (FIGURE 1A) was highly expressed in embryonic stem cells and stringently repressed upon differentiation.
  • Tert h/+ mice were obtained and mated with Tert +I ⁇ mice, generating mice of TerT-’ ⁇ , Tert +I ⁇ , and Tert M+ genotypes.
  • telomerase activity was readily detected in the majority of adult tissues in a Tert +I ⁇ mouse, yet it was present in a limited number of tissues in its Tert h ' ⁇ littermate.
  • FIG. 1C A direct comparison of mTert and hmTert mRNAs in a TerT mh mouse showed that mTert mRNA was expressed in most organs, whereas high hmTert mRNA expression was found only in thymus (FIGURE 1C). Relatively low levels of hmTert mRNA were detected in testis and ovary, and very low levels in intestine and spleen. The hmTert mRNA expression pattern was virtually identical to that of the hTERT mRNA in human tissues (FIGURE ID), indicating that the developmental regulation of the hmTert gene recapitulated that of hTERT in humans.
  • C57BL/6J mice have an average telomere length of approximately 50 kb.
  • Tert +/h Fl mice were crossbred to Tert + ' ⁇ mice to produce Tert hi- and Tert + ' ⁇ offspring, which were then bred with successive generations of Terr 1- mice (FIGURE 3A).
  • Telomere length of these mice were examined by flow cytometry following fluorescence in situ hybridization (Flow-FISH) and telomere restriction fragment (TRF) analysis by Southern blotting (FIGURE 3B, FIGURE 3C).
  • G6 Terr'- mice produced only one offspring which died prematurely, ultimately ending this breeding strategy.
  • the reduction in telomere length in Tert ’' and Tert hl mice did not negatively impact their overall health and well-being, as evidence by their normal body weight in G6 mice (Figure 3E).
  • telomeres Due to the impact of shorter telomeres on mouse fertility, we analyzed the testes of these mice. As illustrated in FIGURE 3F, FIGURE 3G, FIGURE 3H, Tert -1- mice showed testicular atrophy as well as a progressive loss of germ cells in seminiferous tubules starting from G3 mice and worsening in G4 and G5 mice, as previously reported in telomerase-deficient mice. Tert + ' ⁇ mice also exhibited a low level of testicular defects in G4 and G5, but such defects were absent in Tert 11 '- mice. These data indicate that both mTert and hmTert genes help preserve germ cells in the testis, with hmTert showing a slight advantage in preventing germ cell loss.
  • telomere deficiency can impact mouse survival and lifespan.
  • Our results align with those findings, as shown in FIGURES 31, 3 J, G4 and G5 Terr 1- mice had a significantly shortened lifespan, with median survival of approximately 440 and 320 days, respectively.
  • telomere dysfunction in Tert ⁇ z ⁇ mice by the hmTert gene Rescusing telomere dysfunction in Tert ⁇ z ⁇ mice by the hmTert gene [0096]
  • Offspring inherit not only parents’ genotypes but also the lengths of their telomeres.
  • G5 Tert ⁇ ' ⁇ mice displayed severe telomere dysfunction, evidenced by tissue dystrophy, reduced body weight, and a shortened lifespan.
  • the next generation, denoted as G6 Terr'-" 1 and G6 Tert' was generated by crossing G5 Tert ⁇ l ⁇ mice with G5 Tert +! ⁇ and Tert hl ⁇ mice, respectively (FIGURE. 4A).
  • telomere lengths in G6 Tert h ' ⁇ and Tert + ' ⁇ mice were comparable to, or slightly longer than, those of their G5 Terr’- parents and G6 Terr 1 - siblings (FIGURE 4B, FIGURE 5A, and FIGURE 5B).
  • G6 Tert h ' ⁇ and Tert +I ⁇ mice exhibited significantly extended lifespans compared to their G5 Terr'- mice (FIGURE 4C).
  • G5 Terr'- mice died within 383 days, three out of 14 G6 Tert ⁇ / ⁇ h mice survived beyond the entire 460-day experimental period.
  • G6 Terr ⁇ 11 mice had a slightly increased testis weight compared to G6 Terr'-” 1 mice, suggesting that the hmTert gene was functionally similar to, or somewhat better than, the mTert gene for rescuing testicular defects.
  • G6 offspring showed better general health compared to their G5 Terr' ⁇ parents, demonstrating the telomere function-restoring capacities of both the hmTert and mTert genes within a single generation.
  • EXAMPLE 7 hmTert function in immune system
  • G6 Tert +t ⁇ and Tert hl- mice had blood cell counts similar to wildtype mice.
  • the lymphocyte and neutrophil cell percentages within WBCs of G6 Tert -/-h mice were between those of wildtype mice and their G5 Terr 1- parents.
  • G5 Tert -1- mice had a somewhat reduced percentage of CD4 + T cells and dramatically reduced CD8 + T cells, leading to a significant increase of the CD4/CD8 T cell ratio (FIGURE 4G).
  • CD19 + B cells decreased in G5 Terr 1- mice. All these cell counts in G6 Tert +/ ⁇ and Tert h/ ⁇ mice were restored to the levels found in wildtype mice.
  • the gastrointestinal tract is another high proliferation tissue that is affected by telomere dysfunction. Depletion of the intestinal epithelial crypts and severe villus atrophy were observed in small intestines of older G5 Tert -1- (> 8 months), but not in
  • TNF-a another pro- inflammatory cytokine secreted by senescent cells, appeared to be expressed in mouse intestines of all genotypes. Therefore, our data suggest that cellular senescence occurred in Tert ⁇ ' ⁇ intestines with significant telomere dysfunction but was suppressed by the presence of the hmTert gene in this tissue.
  • telomere length setpoint influenced by the hmTert gene.
  • G4 Tert hi mice were continuously intercrossed for 16 generations, from G4.1 to G4.16 (FIGURE 7A).
  • Flow-FISH we monitored telomere length of Tert ! " mice in splenocytes at each generation.
  • FIGURE 7B the average telomere length of Tert M ⁇ mice decreased from 60% to 18% of that observed in wildtype mice from G4 to G4.14, eventually stabilizing at 18-19% in the last three generations (G4.14 to G4.16).
  • both male and female Tert h ' ⁇ mice maintained body weights similar to those of wildtype mice (FIGURE 7C).
  • litter sizes varied, but were largely maintained (FIGURE 7D).
  • Male mice also maintained their testis weight (FIGURE 7E).
  • FIGURE 7F compares the average telomere length of all three genotypes in each generation, from G4.10 to G4.16.
  • telomere length in later generations of mice was also verified using TRF analysis (FIGURE 7G).
  • Two types of telomeres were found in these mice: discrete bands of variable sizes and intensities between 15 and 20kb, and shorter human- like telomere smears.
  • the average lengths of the telomere smear were 8-9 kb in the G4.16 Tert 1 mice and about 7 kb in the Tert M mice. In a G4.14 Tert ⁇ ' ⁇ mouse, the telomere smear was much less apparent.
  • FIGURE 7H shows that, from G4.2 to G4.12, approximately 50% of total born mice were of heterozygous Tert h/ ⁇ genotype, while homozygous Tert h/h and Tert ⁇ ’ ⁇ mice each accounted for about 25% of total progeny, following Mendelian genetics.
  • the few Tert ⁇ ' ⁇ mice that were born were small and die at young ages. These data indicated that short telomeres in late-generation Tert ⁇ ' ⁇ embryos could no longer sustain mouse development.
  • telomeres Maintaining stable human-like telomeres in homozygous Tert h/h mice
  • homozygous Tert h/h offspring from G4, G4.8, and G4.14 were incrossed for 13, 9, and 2 generations, respectively (FIGURE 8A). Average telomere lengths in their progeny were measured by Flow-FISH. The results, depicted in FIGURE 8B, revealed a gradual decrease in telomere length across successive generations. In G4 Tert h/h mice, telomeres decreased from 60% to 30%, while in G4.8 Tert h/h mice, the decline went from 34% to 24%.
  • telomere length in Tert !,/h mice stabilized at a shortened but consistent ranges of 21-24% of wildtype mice, equivalent to an average telomere length of 10-12 kb, similar to reported leukocyte telomere lengths of 9.5 ⁇ 0.7 kb and 10-11 kb in newborn humans.
  • TRF analysis confirmed the presence of discrete telomere bands between 15-20 kb and a human-like telomere smear (FIGURE 8C).
  • telomere smears were 9-10 kb. Regardless of having shortened telomeres, these mice exhibited good health, as demonstrated by stable body weight, litter sizes, and testis weight (FIGURES 8D-8F).
  • Ulcerative colitis is an inflammatory disease associated with telomere shortening and accelerated colon aging in human patients.
  • G4.8h Tert h/h mice remained in good health, and their gastrointestinal tracts appeared normal.
  • cellular proliferation assessed by EdU incorporation, in the colons of G4.8h Tert h/h mice showed a slight decrease, albeit statistically insignificant when compared to wildtype mice.
  • DSS dextran sodium sulfate

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Abstract

La présente divulgation concerne une souris transgénique ainsi que des procédés associés pour une utilisation en tant que modèle de souris. La souris transgénique décrite dans la présente invention comprend un gène de télomérase (hmTert) de souris humanisée contenu dans une lignée germinale murin d'arrière-plan qui fournit des télomères raccourcis qui peuvent répliquer des états humains et divers états de maladie humaine.
PCT/US2024/027191 2023-05-01 2024-05-01 Modèle de souris avec télomère humanisé Pending WO2024229085A2 (fr)

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US20020006913A1 (en) * 1997-11-04 2002-01-17 Von Borstel Reid W. Antimutagenic compositions for treatment and prevention of photodamage to skin
US20030228276A1 (en) * 2002-04-09 2003-12-11 Pitossi Fernando Juan Neuroprotective and neurogenerative effects of the long-term expression of TNF alpha in the substantia nigra and a new animal model for Parkinson's disease
US20140189897A1 (en) * 2011-06-21 2014-07-03 Mayo Foundation For Medical Education And Research Transgenic animals capable of being induced to delete senescent cells
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CA2936488C (fr) * 2014-01-14 2022-05-03 The Medical College Of Wisconsin, Inc. Ciblage de clptm1l pour le traitement et la prevention du cancer
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