A method for constructing a short telomere mouse model

Abstract

The application belongs to the technical field of animal model construction, and discloses a construction method of a short-telomere mouse model. The application obtains a fertilized egg by fertilizing mouse sperm and ovum in vitro, transplants the fertilized egg into a foster mother mouse in vivo for development after culturing the fertilized egg in vitro to the blastocyst stage, and further produces a short-telomere mouse. The application does not need gene editing, has a short modeling cycle, and has reliable and stable effect. By changing the environment of the embryo transplantation period, the embryo telomere extension process is affected to successfully construct a short-telomere offspring model, and the reproduction rate of the mother mouse is not significantly affected. Therefore, the application can provide a construction method of a short-telomere mouse model for exploring the mechanism of telomere shortening and researching aging and other telomere-related phenotypes.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal model construction, and particularly relates to a method for constructing a short telomere mouse model. Background Art

[0002] Telomeres are DNA-protein complexes present at the ends of eukaryotic chromosomes that protect the genome. As cells proliferate, telomeres continue to shorten. When the ends of chromosomes lose the protection of telomeres, the cell apoptosis mechanism is activated. Therefore, telomeres have attracted much attention as biomarkers of aging. Studies have reported an association between short telomeres and cancer, cardiovascular disease, metabolic diseases, and lifespan, but the exact mechanism remains unclear. Therefore, there is an urgent need to establish a stable animal model of short telomeres to promote research on the mechanisms of telomere shortening and telomere-related phenotypes such as aging.

[0003] However, existing animal models of short telomeres are constructed through gene editing technology, mainly including Parp - / - or ATM - / - Knockout mice characterized by Tert - / - or Terc - / - Telomerase-deficient mice. The stability of some gene knockout mouse models is still controversial. - / - Taking mice as an example, one study found that Parp1-deficient mice had shortened telomeres and telomere fusions. However, other studies conducted during the same period did not observe telomere shortening in Parp1-deficient mice, with only mild telomere fusions, suggesting that gene-edited mouse models may be technically unstable. Furthermore, gene-knockout mouse models are difficult to create. In addition to requiring mature gene editing technology, these mouse models require several generations of culture to obtain a stable genotype, which prolongs the mouse modeling cycle.

[0004] Therefore, constructing a short telomere mouse model with stable effects, short modeling cycle, simple modeling method and no changes other than telomeres to the mouse genome will surely be a powerful impetus for exploring the mechanism of telomere shortening and studying telomere-related phenotypes such as aging. It can also provide a suitable animal model for interventional studies on telomere shortening. Summary of the Invention

[0005] The purpose of the present invention is to address the above technical issues and provide an effective, reliable, and simple method for constructing a short telomere mouse model. This method does not rely on gene editing technology and constructs a short telomere mouse model by interfering with the natural telomere elongation process.

[0006] Telomere length is usually shortened with age, but under the action of mechanisms such as homologous recombination and telomerase, the biological process of telomere lengthening is accompanied by the early development of embryos. The present application finds that when the embryo is cultured in vitro to the blastocyst stage, the telomere length of the offspring mice is significantly shortened, that is, the length of the telomere is closely related to the development environment of the embryo. Based on this, the present application attempts to culture mouse embryos in vitro to the blastocyst stage, and then transplant them into the mother mouse to interfere with the early telomere lengthening process of the embryo, so as to construct a short-telomere mouse model.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] A method for constructing a short-telomere mouse model, comprising: fertilizing mouse sperm and oocytes in vitro to obtain zygotes, culturing the zygotes in vitro to the blastocyst stage; transplanting into a surrogate mother mouse for development, and then producing short-telomere mice.

[0009] As a preferred embodiment of the present application, the mouse is an SPF grade mouse of each strain (for example, an ICR strain mouse).

[0010] As a preferred embodiment of the present application, the sperm is added to a conventional commercial sperm capacitation solution (for example, TYH sperm capacitation solution, manufacturer: Aibio, item number: M2050) for 1 hour before fertilization to capacitate the sperm, and the culture condition is a temperature of 37±1℃ and a carbon dioxide content of 4-7%, preferably a temperature of 37℃ and a carbon dioxide content of 5%.

[0011] As a preferred embodiment of the present application, the cumulus-oocyte complex COCs are introduced into a conventional commercial fertilization solution droplet (for example, HTF fertilization solution, manufacturer: Aibio, item number: M1150) before fertilization, and placed in a culture box, and the culture condition is a temperature of 37±1℃ and a carbon dioxide content of 4-7%, preferably a temperature of 37℃ and a carbon dioxide content of 5%.

[0012] As a preferred embodiment of the present application, the in vitro fertilization method comprises intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF).

[0013] As a further preferred embodiment of the present application, 1-3 μl of sperm suspension is sucked from the outer edge of the sperm capacitation solution droplet and injected into the fertilization solution droplet containing the COCs, and the in vitro fertilization dish is placed in a culture box for culture.

[0014] As a further preferred embodiment of the present application, the in vitro culture conditions comprise a conventional commercial cleavage embryo culture medium (e.g. Cleavage Medium, manufacturer: Cook, product number: K-SICM-20, for embryos from zygote to 8-cell stage), a conventional commercial blastocyst culture medium (e.g. Blastocyst Medium, manufacturer: Cook, product number: K-SIBM-20, for embryos from 8-cell stage to blastocyst), and the culture conditions are temperature 37±1°C, carbon dioxide content 4-7%, preferably temperature 37°C, carbon dioxide content 5%.

[0015] As a further preferred embodiment of the present application, the in vitro culture to blastocyst stage is for about 3-4 days.

[0016] The use of in vitro fertilization in preparing a short-telomere mouse model, characterized in that the fertilized eggs are cultured in vitro to the blastocyst stage and then transplanted into a surrogate mother mouse for development, and the resulting mouse is a short-telomere mouse.

[0017] As an embodiment of the present application, a method for constructing a short-telomere mouse model comprises the following steps:

[0018] (1) Preparation of sperm: pick up the paste-like sperm into another sperm capacitation medium droplet, and place the sperm capacitation dish into the incubator for 1 hour for sperm capacitation.

[0019] (2) Preparation of oocytes: take the cumulus-oocyte complex (COCs) released in mineral oil. Introduce the COCs into a 100 μl fertilization medium droplet with an ophthalmic forceps, and place it into the incubator for the sperm capacitation process, with the culture conditions being temperature 37°C and carbon dioxide content 5%.

[0020] (3) In vitro fertilization: inject 1-3 μl of sperm suspension from the outer edge of the sperm capacitation medium droplet into the fertilization medium droplet containing the COCs. Place the in vitro fertilization dish into the incubator for about 6 hours for in vitro fertilization, with the culture conditions being temperature 37°C and carbon dioxide content 5%.

[0021] (4) Embryo culture and transplantation: Prepare pseudopregnant surrogate female mice when in vitro fertilization. Prepare the cleavage embryo culture dish, 100ul of conventional commercial cleavage embryo culture solution (such as cleavage culture solution, manufacturer: Cook, product number: K-SICM-20), cover with mineral oil, and put into the incubator in advance for 6 hours. After in vitro fertilization for 6 hours, wash the fertilized eggs with in vitro fertilization solution for three times, observe the male and female pronuclei after washing, remove the unfertilized eggs, transfer the fertilized eggs into the cleavage embryo culture solution, and put the cleavage embryo culture dish into the incubator for culture, the culture condition is 37℃ and 5% carbon dioxide content. After about 24 hours of culture, observe the embryo state, remove the development blocked embryos, and keep the normal development to 2-cell stage embryos for further culture for about 24 hours. Then prepare the blastocyst culture dish, 100ul of conventional commercial blastocyst culture solution (such as blastocyst culture solution, manufacturer: Cook, product number: K-SIBM-20), cover with mineral oil, and put into the incubator in advance for 6 hours. Observe the embryo development, remove the development blocked embryos, select the normal development to 8-cell stage embryos, transfer into the blastocyst culture solution, and put into the incubator. After about 24 hours of culture, observe the embryo development, remove the development blocked embryos, select the normal development to blastocyst stage embryos, and transplant into the uterus of surrogate female mice, 15 blastocysts per female mouse, and feed in single cage for delivery. The short telomere mouse is obtained.

[0022] The present application does not protect the method of obtaining sperm and eggs from mice. Only the modeling method of in vitro fertilization and development to blastocyst using the just obtained sperm and eggs, and transplantation into the body of surrogate mother mice for development to produce short telomere mice is protected.

[0023] After the surrogate female mice deliver, the peripheral blood, heart, liver, brain, lung, kidney and intestinal tissue of the mice on the first day after birth are detected for relative telomere length, and the results show that the telomere length of the mice in each tissue is significantly shortened. The tail vein blood of the mice at six months after birth is detected for telomere length, and the results also show significant shortening. According to the construction method of the present application, the short telomere mouse model can be successfully constructed.

[0024] The present application has the following beneficial effects:

[0025] The short telomere mouse model constructed by the present application has the following advantages:

[0026] (1) The modeling method of the mouse model constructed by the present application is simple, the modeling period is short, and no gene editing is needed. By changing the environment during embryo transplantation, the extension process of the embryo telomere is affected, and the short telomere offspring is caused. This process has no significant effect on the reproduction rate of the mother mice. Therefore, the present application provides an important mouse model construction method for exploring the mechanism of telomere shortening and studying aging and other telomere related phenotypes.

[0027] (2) The mouse model constructed by the application has stable and reliable modeling effect, and the telomere length of the peripheral blood, heart, liver, brain, lung, kidney and intestinal tissue of the mouse constructed by the model is significantly shortened.

[0028] (3) The short telomere mouse model constructed based on the application can be used to simulate the embryo transfer process of the human assisted reproduction process, and is helpful to explore the mechanism of telomere shortening and carry out intervention research on short telomere. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The short telomere mouse construction scheme provided by the application is shown in the figure.

[0030] Figure 2 The telomere length comparison diagram of the peripheral blood of the short telomere mouse and the control mouse on the first day after birth provided by the embodiment of the application is shown in the figure.

[0031] Figure 3 The telomere length comparison diagram of the brain tissue of the short telomere mouse and the control mouse on the first day after birth provided by the embodiment of the application is shown in the figure.

[0032] Figure 4 The telomere length comparison diagram of the heart tissue of the short telomere mouse and the control mouse on the first day after birth provided by the embodiment of the application is shown in the figure.

[0033] Figure 5 The telomere length comparison diagram of the liver tissue of the short telomere mouse and the control mouse on the first day after birth provided by the embodiment of the application is shown in the figure.

[0034] Figure 6 The telomere length comparison diagram of the kidney tissue of the short telomere mouse and the control mouse on the first day after birth provided by the embodiment of the application is shown in the figure.

[0035] Figure 7 The telomere length comparison diagram of the intestinal tissue of the short telomere mouse and the control mouse on the first day after birth provided by the embodiment of the application is shown in the figure.

[0036] Figure 8 The telomere length comparison diagram of the lung tissue of the short telomere mouse and the control mouse on the first day after birth provided by the embodiment of the application is shown in the figure.

[0037] Figure 9 The telomere length comparison diagram of the peripheral blood of the short telomere mouse and the control mouse on the first day after birth provided by the embodiment of the application is shown in the figure. DETAILED DESCRIPTION

[0038] Embodiment 1

[0039] The embodiment provides a construction method of a short telomere mouse model, and the flow is as follows:

[0040] (1) Sperm preparation: The donor male mice were housed individually one week before sperm collection. Thirty minutes before sperm collection, two 100 μΐ droplets of conventional commercial sperm capacitation medium (e.g., TYH sperm capacitation medium, manufacturer: Aibio, catalog number: M2050) were prepared in a sperm capacitation dish and overlaid with mineral oil. One 100 μΐ droplet of conventional commercial in vitro fertilization medium (e.g., HTF fertilization medium, manufacturer: Aibio, catalog number: M1150) was prepared in an in vitro fertilization dish and overlaid with mineral oil, and both were equilibrated in a 37 °C, 5% CO2 incubator. The 8-12 week old male mice were sacrificed by cervical dislocation, the abdominal cavity was cut open, and the cauda epididymidis was removed and placed on a sterile filter paper to remove blood, fat, and other impurities. The cauda epididymidis was then placed in the sperm capacitation medium droplet in the sperm capacitation dish, and the sperm was squeezed out. The sperm was then picked up in another sperm capacitation medium droplet. The sperm capacitation dish was placed in the incubator for 1 hour to allow sperm capacitation, and the incubation conditions were 37 °C and 5% CO2.

[0041] (2) Oocyte preparation: The 4-5 week old female mice were superovulated by intraperitoneal injection of pregnant mare serum gonadotropin (PMSG) at a dose of 5 IU per mouse. Forty-eight hours after PMSG injection, human chorionic gonadotropin (HCG) was injected at a dose of 5 IU per mouse. Fifteen hours after HCG injection, the female mice were sacrificed by cervical dislocation, the abdominal cavity was cut open, and the oviducts were removed and placed in mineral oil in the in vitro fertilization dish. The oviducts were cut and squeezed to release the cumulus-oocyte complexes (COCs) into the mineral oil. The COCs were introduced into the 100 μΐ droplet of fertilization medium using an ophthalmic forceps, and the in vitro fertilization dish was placed in the incubator. The incubation conditions were 37 °C and 5% CO2.

[0042] (3) In vitro fertilization: 1-3 μΐ of the sperm suspension was pipetted from the outer edge of the sperm capacitation medium droplet and injected into the droplet of fertilization medium containing the COCs. The in vitro fertilization dish was placed in the incubator, and the in vitro fertilization was performed for about 6 hours. The incubation conditions were 37 °C and 5% CO2.

[0043] (4) Embryo culture and transfer: Prepare pseudopregnant surrogate female mice at the time of in vitro fertilization. Prepare embryo culture dishes, 100 μl of conventional commercial embryo culture solution (e.g., embryo culture solution, manufacturer: Cook, product number: K-SICM-20), overlaid with mineral oil, and place in the incubator for 6 hours in advance. After 6 hours of in vitro fertilization, wash the fertilized eggs in the in vitro fertilization solution three times, observe the male and female pronuclei after washing, remove the unfertilized eggs, transfer the fertilized eggs into the embryo culture solution, and place the embryo culture dishes in the incubator for culture. The culture conditions are a temperature of 37°C and a carbon dioxide content of 5%. After about 24 hours of culture, observe the state of the embryos, remove the developmentally arrested embryos, and retain the normally developed 2-cell stage embryos. After about 24 hours of further culture, prepare blastocyst culture dishes, 100 μl of conventional commercial blastocyst culture solution (e.g., blastocyst culture solution, manufacturer: Cook, product number: K-SIBM-20), overlaid with mineral oil, and place in the incubator for 6 hours in advance. Observe the state of the embryo development, remove the developmentally arrested embryos, and select normally developed 8-cell stage embryos. Transfer the selected embryos into the blastocyst culture solution and place in the incubator. After about 24 hours of culture, observe the state of the embryo development, remove the developmentally arrested embryos, select normally developed blastocyst stage embryos, and transfer the selected embryos into the uterus of surrogate female mice. Transfer 15 blastocysts into the uterus of each female mouse, and raise the mice individually in cages until delivery. Obtain short-telomere mice.

[0044] (5) Comparative experiment: the experiment is divided into two groups, group A is to use the modeling method described above, group B is to transplant into the surrogate female mouse unilateral oviduct when the fertilized egg develops to 2-cell stage (the method is the same as above) (because it cannot be guaranteed that the mice come from the same parents, there is a genetic factor to interfere with the length of telomeres, and there is no normal in-vivo fertilized mouse control), 15 2-cell stage embryos are transplanted into each female mouse, and they are fed singly in a single cage until the delivery of the obtained mice. Part of the mice in groups A and B are executed by decapitation on the day of delivery, and peripheral blood, heart, liver, brain, lung, kidney, intestinal tissue are obtained by dissection, DNA is extracted, and qPCR method is used to detect the relative telomere length (RTL) of each tissue. In addition, the remaining mice in the two groups are fed until they are six months old, and the tail vein blood of the mice in the two groups is taken, DNA is extracted, and qPCR method is used to detect RTL. The detection method is to use two pairs of primers to amplify the DNA template respectively: (1) Tel-F primer sequence: 5 'CGG TTT GTTTGG GTT TGG GTT TGG GTT TGG GTT TGG GTT 3'(300 nM), Tel-R primer sequence: 5 'GGC TTG CCTTAC CCT TAC CCT TAC CCT TAC CCT TAC CCT 3'(300 nM), reaction conditions: 95℃, 10min; 30 cycles of 95℃, 15s, then 56℃, 1min. Single reaction system: 5μl ChamQ SYBR qPCR Master Mix (manufacturer: Vazyme, product number: Q331-02), F, R primers (concentration as described above), 20ng DNA template, ddH2O to 10μl. (2) 36B4-F primer sequence: 5 'GTT GGG AGT TGG ACT ATG GAC 3'(300 nM), 36B4-R primer sequence: 5 'TGAACT GAT TGG ACA CAC ACA 3'(500 nM), reaction conditions: 95℃, 10min; 35 cycles of 95℃, 15s, then 52℃, 20s, then 72℃, 30s. Single reaction system: 5μl ChamQ SYBR qPCR Master Mix, F, R primers (concentration as described above), 20ng DNA template, ddH2O to 10μl. According to the CT values of the two reactions of the sample, the RTL of the sample is calculated, and the calculation method is:

[0045]

[0046] Statistical analyses were performed using specialized statistical analysis software (R v4.0.2). (1) Data normalization: Data were logarithmically transformed to ensure normal distribution; z-score transformation was performed on different test batches to ensure data comparability. (2) The chi-square test was used to compare the reproductive rate of female mice between groups; the Student's t-test was used to compare the telomere length of mice between groups. The statistical significance level was set at 0.05, and all statistical tests were two-sided.

[0047] The results showed no significant difference in reproductive rate between the female mice in Groups A and B. On the first day after birth, telomere lengths in seven different tissues, including peripheral blood, heart, liver, brain, lung, kidney, and intestine, were significantly shorter in Group A than in Group B. Even after reaching adulthood (six months of age), peripheral blood telomere lengths in Group A remained significantly shorter than those in Group B. These results demonstrate that the short telomere mouse model was successfully established using the methods of the present invention.

Claims

1. A method for constructing a short telomere mouse model, characterized in that: include: Mouse sperm and eggs are fertilized in vitro to obtain fertilized eggs, which are then cultured in vitro to the blastocyst stage; the fertilized eggs are then transplanted into surrogate mother mice for development, thereby producing short-telomere mice; wherein, before fertilization, sperm is added to sperm capacitation solution and cultured for 50-70 minutes to capamate the sperm, the culture conditions being a temperature of 37±1°C and a carbon dioxide content of 4-7%, the cumulus ovary-oocyte complex (COCs) is introduced into a microdroplet of fertilization solution, and the microdroplet is placed in an incubator, the culture conditions being a temperature of 37±1°C and a carbon dioxide content of 4-7%; the in vitro fertilization method comprises aspirating 1-3 μl of sperm suspension from the outer edge of the microdroplet of sperm capacitation solution and injecting it into a microdroplet of fertilization solution containing COCs, and the in vitro fertilization dish is placed in an incubator for culture, the culture conditions being a temperature of 37±1°C and a carbon dioxide content of 4-7%.

2. The method for constructing a short telomere mouse model according to claim 1, characterized in that: The mice are SPF grade mice of various strains.

3. The method for constructing a short telomere mouse model according to any one of claims 1-2, characterized in that: The culture medium used for 2-cell embryos to 8-cell embryos is conventional commercial cleavage embryo culture medium, and the culture medium used for 8-cell embryos to blastocysts is conventional commercial blastocyst culture medium.

4. The method for constructing a short telomere mouse model according to claim 1, wherein: The in vitro culture to the blastocyst stage is 3-4 days.

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