Construction method and application of mouse testis tissue stress granule animal model

Abstract

The application belongs to the technical field of animal models, and provides a method for constructing a mouse testis tissue stress granule animal model, which comprises the following steps: after the mouse is anesthetized, the testis and epididymis of one side of the mouse are taken out; a sodium arsenite solution containing fast green dye is injected into the testis of the mouse through the entrance of the rete testis, and the concentration of the sodium arsenite solution is 30-50 mM; the wound of the mouse is sutured, the mouse is killed after the mouse recovers for 3 hours, and the testis tissue is separated for an immunofluorescence experiment; when obvious stress granules are formed, it is indicated that the model construction is successful. The application also provides an application of the method for constructing an animal model in identifying the generation of stress granules of spermatocytes and in identifying the generation of RNA binding protein SERBP1. The application provides a method for exploring the role of SERBP1 protein in the stress granules of the mouse testis tissue, a related mechanism and a possible molecular pathway, and provides a new theoretical basis for guiding the generation and recovery of cell stress granules under special conditions.

Description

Technical Field

[0001] This invention belongs to the field of animal model technology, and particularly relates to a method for constructing and applying an in vivo stress particle animal model. Background Technology

[0002] The RNA-binding protein SERBP1 is predicted to bind to the SUMO domain, mRNA 3'-UTR, and ribosomes. It is predicted to participate in the regulation of tissue and apoptosis processes in PML bodies, to be localized in the cytosol and plasma membrane, and to have enhanced activity in the cytoplasm and nucleus. SERBP1 is also predicted to generate stress granules under specific environments, such as high pressure, hypoxia, acid-base imbalances, and toxins, and to co-localize with stress granule proteins TIAR and G3BP1.

[0003] Currently, there is very little research on SERBP1, especially on the generation of stress granules in testicular tissue, and its generation mechanism is unclear. Therefore, it is of great significance to establish a mouse animal model of stress granules in testicular tissue and explore the role, related mechanisms, and possible molecular pathways of SERBP1 protein in stress granules in mouse testicular tissue. At the same time, it will provide new theoretical basis for guiding the generation and recovery of cellular stress granules under special conditions. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing a mouse testicular tissue stress particle animal model, aiming to solve the problems existing in the background art.

[0005] The present invention is implemented as follows: a method for constructing a mouse testicular tissue stress granulation animal model includes the following steps: After anesthetizing the mouse, the testis and epididymis on one side of the mouse were removed; A sodium arsenite solution containing Fast Green dye was injected into the mouse testes via the testicular reticulum inlet. The concentration of the sodium arsenite solution was 30-50 mM. The mouse wound was sutured, and the mouse was allowed to recover. After 3 hours, the mouse was sacrificed, and the testicular tissue was separated, sectioned, and subjected to immunofluorescence experiments. The presence of obvious stress granules indicated that the model was successfully constructed.

[0006] Preferably, in the step of anesthetizing the mice, 50 mg of 1% pentobarbital per 1 kg of mouse body weight is injected intraperitoneally.

[0007] Preferably, the step of removing one side of the mouse's testis and epididymis specifically includes: making a transverse incision 1 cm above the pubic symphysis in the surgical area of ​​the mouse, pulling out one side of the mouse's testis and epididymis, placing the testis and epididymis on gauze, and transferring them to a stereomicroscope.

[0008] Preferably, in the step of injecting a sodium arsenite solution containing Fast Green dye into the mouse testes via the testicular reticulum inlet, the Fast Green dye concentration is 0.5% and is prepared from a 0.01M phosphate buffer solution; The injection angle is 25-35°; The injected liquid volume was 1 μL sodium arsenite per 3 g mouse body weight.

[0009] Preferably, in the step of euthanizing the mouse after waiting 3 hours, the mouse is euthanized by neck dislocation.

[0010] Preferably, when testicular tissue sections show obvious stress granule formation and co-localization of DZAL green fluorescent protein and the stress granule protein marker TIAR, it is a positive spermatocyte stress granule model group. When testicular tissue sections show obvious stress granule formation and co-localization of SERBP1 red fluorescent protein and stress granule protein marker G3BP1, it is a positive SERBP1 stress granule model group.

[0011] Another objective of this invention is to provide a method for constructing a mouse testicular tissue stress granule animal model and its application in identifying stress granules produced by spermatocytes.

[0012] Another objective of this invention is to provide a method for constructing a mouse testicular tissue stress granule animal model and its application in recognizing the generation of RNA-binding protein SERBP1.

[0013] This invention provides a method for constructing a mouse testicular stress granulation animal model. Based on microscopic injection technology, an appropriate concentration of sodium arsenite solution is injected, successfully constructing an in vivo stress granulation animal model. Verification shows that unilateral injection of 10 μL of 30-50 mM sodium arsenite into one testis of mice can induce significant stress in the mouse testicular tissue. Molecular biology, immunofluorescence, and other techniques are used to study the role, related mechanisms, and possible molecular pathways involved of the SERBP1 protein in mouse testicular stress granulations, providing new theoretical basis for understanding how sodium arsenite-induced stress affects the development of testicular spermatogenic cells. Attached Figure Description

[0014] Figure 1 A schematic diagram illustrating a method for constructing and analyzing a mouse testicular tissue stress granulation animal model provided in an embodiment of the present invention; Figure 2 This is an image showing the effect of injecting sodium arsenite into the testes of mice according to an embodiment of the present invention. Figure 3 This is a comparison of testicular size in mice 3 hours after surgery, provided in an embodiment of the present invention. Figure 4This is a colocalization map of DZAL green fluorescent protein and the stress particle protein marker TIAR provided in an embodiment of the present invention; Figure 5 This is a colocalization map of SERBP1 red fluorescent protein and stress granule protein marker G3BP1 provided in an embodiment of the present invention. Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0016] A method for constructing a mouse testicular tissue stress granulation animal model, specifically including the following steps: (1) Prepare adult male C57 mice and 1% pentobarbital anesthetic before the experiment. Five minutes before the dissection, inject 50 mg of 1% pentobarbital into the peritoneum for every 1 kg of body weight of the mice. (2) After the mice are anesthetized, the surgical area instruments are prepared and disinfected with alcohol. The surgical area of ​​the mice is a transverse incision 1 cm above the pubic symphysis. The testis and epididymis of one side of the mice are pulled out, and the testis and epididymis are placed on gauze and transferred to a stereomicroscope. (3) Determine the appropriate sodium arsenite concentration gradient (30, 40, 50 mM), and use an injection needle to inject a sodium arsenite solution containing 0.5% Fast Green (prepared from 0.01 M PBS (Phosphate-Buffered Saline)) into the mouse testis through the outflow tube (this is the testicular reticulum inlet). The injection angle is 25-35°, and the angle should be adjusted during the injection process. (4) During the injection of the mouse testes, green liquid can be seen filling the seminiferous tubules (the volume of the injected liquid is about 10 μL / 3g of mouse body weight). After the injection, the mouse testes can be seen to turn green. (5) After the injection, carefully suture the abdominal incision of the mouse and place the mouse on a warming blanket. At the same time, observe the mouse's condition, including respiratory rate and body temperature. After the mouse recovers, put it back in the cage and wait for 3 hours. (6) After waiting for 3 hours, the mice were euthanized by neck dislocation, the testicular tissue was separated, fixed, frozen and sectioned (section thickness was 5 micrometers), and then the subsequent immunofluorescence experiment was performed for verification. The immunofluorescence experiment verified that: if the testicular tissue sections of the sodium arsenite-treated group showed obvious aggregation points of the stress granule marker TIAR protein and co-localized with the spermatocyte marker DAZL, it was a spermatocyte positive induction group, which could be used to identify the production of stress granules by spermatocytes and study the effect of stress granule formation on spermatocyte development. If testicular tissue sections from the sodium arsenite-treated group show obvious stress granule formation and co-localize with RNA-binding protein SERBP1, it is considered a SERBP1-positive induction group. This can be used to identify the production of RNA-binding protein SERBP1 and study the effect of RNA-binding protein SERBP1 on stress granule formation. The basic information of SERBP1 is as follows: Gene name (Ensembl): Ensembl:ENSMUSG00000036371; Gene ID: 66870; The gene sequence is shown in the sequence listing SEQ ID NO:1.

[0017] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0018] like Figure 1 As shown, based on microinjection technology, a sodium arsenite solution containing Fast Green dye, prepared in vitro, was injected into mouse testicular tissue through an output tube to construct an in vivo stress granulation animal model. The steps are as follows: (1) Before the experiment, C57 male mice were injected intraperitoneally with 1% pentobarbital anesthetic (50 mg / kg body weight). (2) Dissect the mouse, remove one side of the testis and epididymis, place them on gauze, and transfer them to a stereomicroscope; (3) Using an injection needle, the sodium arsenite solution containing the Fast Green dye was injected into the mouse testis through the outflow tube (which is the testicular reticulum inlet). The injection angle was 25-35°. During the injection, the angle was adjusted. The injection concentration gradient of sodium arsenite is shown in Table 1. Table 1

[0019] (4) During the testicular injection, observe the green liquid filling the seminiferous tubules. The volume of the injected liquid is about 10 μL (1 μL sodium arsenite / 3 g mouse body weight). After the injection, the mouse testes will turn green, indicating that the injection was successful. like Figure 2 The image shown is an illustration of the effect of testicular injection in an in vivo stress particle animal model constructed through the example. (5) Three hours after the testes were injected with sodium arsenite, the testicular tissue of the mice described above was photographed. Figure 3 As shown, no significant changes were found in the testicular tissue of the stress granule model group, indicating that sodium arsenite injection has no effect on the morphology and size of testicular tissue, but it can induce the seminiferous tubules to produce stress granules. (6) Immunofluorescence staining of frozen sections of the above-mentioned mouse testicular tissue revealed that DZAL green fluorescent protein co-localized with the stress granule protein marker TIAR, indicating a positive spermatocyte stress granule model group. Figure 4 As shown; the presence of SERBP1 red fluorescent protein co-localization with the stress granule protein marker G3BP1 indicates a positive SERBP1 stress granule model group, as shown. Figure 5 As shown.

[0020] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a mouse testicular tissue stress granulation animal model, characterized in that, Includes the following steps: After anesthetizing the mouse, the testis and epididymis on one side of the mouse were removed; A sodium arsenite solution containing Fast Green dye was injected into the mouse testes via the testicular reticulum inlet. The concentration of the sodium arsenite solution was 30-50 mM. The mouse wound was sutured, and the mouse was allowed to recover. After 3 hours, the mouse was sacrificed, and the testicular tissue was separated, sectioned, and subjected to immunofluorescence experiments. The presence of obvious stress granules indicated that the model was successfully constructed. When testicular tissue sections show obvious stress granule formation and co-localization of DZAL green fluorescent protein and the stress granule protein marker TIAR, it is a positive spermatocyte stress granule model group. When testicular tissue sections show obvious stress granule formation and co-localization of SERBP1 red fluorescent protein and stress granule protein marker G3BP1, it is a positive SERBP1 stress granule model group.

2. The method for constructing a mouse testicular tissue stress granulation animal model according to claim 1, characterized in that, In the process of anesthetizing mice, 50 mg of 1% pentobarbital per 1 kg of mouse body weight was injected intraperitoneally.

3. The method for constructing a mouse testicular tissue stress granulation animal model according to claim 1, characterized in that, The steps for removing one side of the mouse's testis and epididymis include: making a transverse incision 1 cm above the pubic symphysis in the surgical area of ​​the mouse, pulling out one side of the mouse's testis and epididymis, placing the testis and epididymis on gauze, and transferring them to a stereomicroscope.

4. The method for constructing a mouse testicular tissue stress granulation animal model according to claim 1, characterized in that, In the step of injecting a sodium arsenite solution containing Fast Green dye into the mouse testes via the testicular reticulum inlet, the Fast Green dye concentration is 0.5% and is prepared from a 0.01M phosphate buffer solution; The injection angle is 25-35°; The injected liquid volume was 1 μL sodium arsenite per 3 g mouse body weight.

5. The method for constructing a mouse testicular tissue stress granulation animal model according to claim 1, characterized in that, In the step of euthanizing the mice after waiting 3 hours, the mice were euthanized by cervical dislocation.

6. The application of a method for constructing a mouse testicular tissue stress granule animal model as described in any one of claims 1-5 in identifying stress granules produced by spermatocytes.

7. The application of a method for constructing a mouse testicular tissue stress particle animal model as described in any one of claims 1-5 in recognizing the generation of RNA-binding protein SERBP1.

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