Construction method of mouse placenta-targeted gene interference model and application thereof

Abstract

The application discloses a kind of construction methods of mouse placenta targeted gene interference model and its application.The method comprises the following steps: first, pregnant mouse is anaesthetized and abdominal operation is carried out, and the uterus containing placenta is exposed;Then, the adeno-associated virus carrying gene interference element is directly injected into target placental tissue;Finally, the wound is sutured to complete model construction.The present application overcomes the problem of low virus delivery efficiency caused by maternal-fetal barrier by intraoperative direct site injection, and realizes the efficient and specific gene interference of placental tissue.The model constructed is targeted and precise, has high interference efficiency and low off-target effect, and provides an effective tool for studying placental gene function and pregnancy-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of experimental animal model construction technology, specifically relating to a method for constructing a placental-targeted gene interference animal model by intraoperative targeted injection of adeno-associated virus, and its application in the study of pregnancy-related diseases. Background Technology

[0002] The maternal-fetal interface is a crucial biological interface for the exchange of substances, immune regulation, and signal communication between the mother and fetus during pregnancy. It is primarily composed of decidual tissue derived from the mother and chorionic villi derived from the fetus. The integrity of its structure and function is fundamental to maintaining a normal pregnancy, ensuring fetal growth and development, and achieving fetal immune immunity. Abnormalities in maternal-fetal interface function are closely related to the occurrence of various pregnancy-related diseases, such as fetal growth restriction, miscarriage, and preeclampsia. Therefore, targeted intervention of specific genes at the maternal-fetal interface in animal models is crucial for in-depth research into the molecular mechanisms of pregnancy-related diseases.

[0003] Adeno-associated virus (AAV) has become a core tool for in vivo gene delivery and functional studies due to its high safety, broad host range, and persistent expression. In gene interference studies, short hairpin RNA (shRNA) and other interfering sequences are typically loaded into recombinant AAV (rAAV) vectors to inhibit the expression of target genes by infecting target cells. Currently, the conventional routes for in vivo AAV injection mainly include tail vein injection, intraperitoneal injection, and local tissue injection. Among these, intravenous or intraperitoneal injection relies on systemic blood circulation to deliver the virus to the target organ, requiring not only high viral titers but also easy accumulation in non-target organs such as the liver, leading to off-target effects and potential toxicity. Although targeting can be improved to some extent through tissue-specific promoters or the Cre-LoxP system, these methods are complex, costly, and their delivery efficiency remains limited for tissues with special barriers.

[0004] The maternal-fetal interface is a unique organ with multiple barriers. Its unique physical structure (such as the placental barrier) and immune microenvironment effectively prevent large molecules, pathogens, and immune cells from the maternal bloodstream from entering the fetal side, thus ensuring pregnancy safety. However, this protective mechanism also poses a major technical obstacle to the efficient and specific delivery of gene manipulation tools such as AAV to placental tissue. Existing research shows that AAV, administered via tail vein or intraperitoneal injection in mice, has difficulty effectively crossing the maternal-fetal barrier, resulting in less than ideal infection efficiency and gene interference effects in placental tissue. This severely restricts the development of in vivo placental-specific gene function research using AAV vectors.

[0005] Although local injection of AAV technology has been successfully applied in targeted studies of organs such as the nervous system, the placenta's unique anatomical and physiological characteristics—its location deep within the abdominal cavity, its connection to multiple embryos, and its rich blood supply making it prone to bleeding—make precise and safe operation difficult with traditional local injection methods (such as superficial puncture). Currently, there are no mature, efficient, and systematically reported intraoperative placental targeted AAV injection techniques that ensure the survival rate of pregnant mice and fetuses. Therefore, there is an urgent need in this field to develop a feasible, highly efficient, targeted, and minimally impactful method for constructing placental targeted gene interference models to fill this technological gap. Summary of the Invention

[0006] This invention aims to overcome the technical obstacles in existing placental tissue-targeted gene interference, such as low efficiency, poor specificity, and complex operation, and provides a simple, efficient, and highly accurate method for constructing a mouse placental-targeted gene interference model and its related applications.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: The first aspect of this invention provides a method for constructing a mouse placental-targeting gene interference model, comprising the following steps: Pregnant mice were anesthetized and underwent abdominal surgery to expose the uterus containing the target placenta; Adeno-associated viruses carrying gene interference elements are directly injected into the target placental tissue; Suture the wound.

[0008] In a preferred embodiment, the step of exposing the uterus and placenta includes: making an incision in the abdomen of a pregnant mouse to open the abdominal cavity, and exposing the uterus and placenta located in the posterior part of the abdominal cavity after the intestines are opened.

[0009] In one specific implementation, the injection is performed via microinjection.

[0010] In one specific embodiment, the serotype of the adeno-associated virus is selected from one of AAV2, AAV9, or AAV2 / 9.

[0011] The adeno-associated virus (AAV) mentioned refers to a recombinant AAV vector, a small, non-enveloped single-stranded DNA virus. It is characterized by high safety, low immunogenicity, and the ability to mediate the long-term stable expression of exogenous genes, and is a commonly used gene delivery tool in gene therapy and functional research.

[0012] The gene interference element refers to a genetic sequence loaded into a viral vector to specifically inhibit or reduce the expression of a target gene. Its mechanism of action includes, but is not limited to, RNA interference and CRISPR interference.

[0013] Microinjection refers to a technique that uses ultra-fine needles for precise injection with the assistance of optical magnification equipment such as stereomicroscopes. This technique enables the precise delivery of minute amounts of liquid, minimizing tissue damage.

[0014] The serotype refers to a specific type of adeno-associated virus (AAV) capsid protein; different serotypes exhibit varying infectivity to tissues and cells. AAV2 / 9 is a chimeric serotype of AAV2 and AAV9, possessing the tissue tropism advantages of both.

[0015] In one specific implementation, the gene interference element is a short hairpin RNA.

[0016] The short hairpin RNA is an RNA molecule with a stem-loop structure that can be processed into small interfering RNA within cells. It specifically degrades the mRNA of target genes through RNA interference mechanisms, thereby inhibiting gene expression.

[0017] In one specific implementation, the pregnant mouse is in the embryonic stage from day 12.5 to day 15.5.

[0018] The period from day 12.5 to day 15.5 of the embryonic period refers to the number of days counted from the first thrombus observed in the mouse, usually denoted by E. During this stage, the placenta is relatively mature, its size is suitable for surgical manipulation, and the fetal survival rate is high.

[0019] In one specific implementation, the injection target site is the labyrinthine region of the placenta.

[0020] The labyrinthine region refers to the placental labyrinth structure in the mouse placenta, which is rich in various trophoblast cells, blood vessels and endothelial cells, and is an important site for material exchange and signal transduction between the mother and fetus.

[0021] In one specific implementation, during the injection step, the viral load injected into each placenta is 1 × 10⁻⁶. 10 vg -5×10 10 vg, injection volume is 1-5 μL.

[0022] The term vg refers to the viral genome copy number, a unit used to represent the dosage of viral particles.

[0023] The second aspect of the present invention provides a mouse placental-targeting gene interference model constructed by the method described in the first aspect of the present invention.

[0024] The third aspect of the present invention provides the application of the method described in the first aspect of the present invention in the preparation of animal models for studying pregnancy-related diseases.

[0025] The pregnancy-related diseases include, but are not limited to, fetal growth restriction, preeclampsia, gestational diabetes mellitus, recurrent miscarriage, and other diseases related to placental dysfunction.

[0026] The beneficial effects of this invention are as follows: by using intraoperative direct exposure and targeted injection techniques, the limitations of the maternal-fetal barrier on virus delivery are overcome, achieving high concentration enrichment and efficient infection of adeno-associated virus in placental tissue, thereby significantly improving the efficiency and specificity of gene interference; the interference effect of this method is mainly limited to the target placenta, effectively reducing the risk of systemic off-target effects, and the experimental background is clearer; its operation procedure is standardized, parameters are clear, the modeling success rate is high and the reproducibility is good, providing a stable and reliable animal model platform for placental function research and the exploration of the mechanisms of pregnancy-related diseases. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the surgical procedure for placental targeted injection of AAV in Embodiment 1 of the present invention.

[0028] Figure 2 The images show a comparison of immunofluorescence staining of placental tissue infected with viruses under different injection methods in Example 1 of this invention; where (A) is the tail vein injection group and (B) is the placental targeted injection group.

[0029] Figure 3 This is a graph showing the protein and mRNA levels used to verify the gene interference effect in Example 1 of the present invention; wherein, (A) is the Western Blot result of the tail vein injection group and the negative control group, (B) is the Western Blot result of the placental targeted injection group and the negative control group, and (C) is a qPCR quantitative statistical graph of the gene mRNA expression level of the placental targeted injection group and the negative control group. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the present invention clearer, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. All materials and instruments used in the embodiments are commercially available. Experimental methods not specifying specific conditions in the embodiments are generally performed under conventional conditions or according to the manufacturer's recommendations.

[0031] Example 1: Method for constructing a gene interference model by placental-targeted injection of AAV and its effect verification

[0032] 1. Laboratory animals and viruses

[0033] The mice used in the experiment were 6-8 week old female C57BL / 6J mice, purchased from Spiford (Beijing) Biotechnology Co., Ltd., and were housed with male mice of the same lineage. The day when the thrombus was found was counted as day 0.5 of gestation (E0.5).

[0034] The viral vector was recombinant adeno-associated virus (rAAV), driven by the PSCAAV-U6 promoter, with serotype AAV2 / 9 (a chimeric serotype of AAV2 and AAV9), and a viral titer of 5 × 10⁻⁶. 12 vg / mL.

[0035] 2. Experimental Grouping

[0036] Pregnant C57BL / 6J mice at day 13.5 of gestation (E13.5) were randomly divided into two groups (each group containing at least 5 mice): (1) Experimental group (placental targeted injection group, i.e., placental targeted injection of aav-sh-target gene): The uterus and placenta were exposed by surgery, and the virus was injected at a specific site. The injection volume was 2.5 x 10 10 vg / placenta (a pregnant mouse pregnancy produces approximately 10 fetuses and a placenta); (2) Method control group (tail vein injection group, i.e., tail vein injection of aav-sh-target gene): The same virus was injected into the tail vein of mice, with a total injection volume of 3.0 × 10 11 vg; (3) Negative control group (placental injection of aav-NC): AAV carrying disordered shRNA was injected into the placenta at a specific site.

[0037] 3. Placental targeted injection surgery procedure

[0038] The experimental group strictly followed the procedures below (e.g.) Figure 1 (as shown) (1) Preoperative preparation: The hair on the abdomen of the mice was shaved off using a hair removal device. The pregnant mice were anesthetized by injecting 20 ml / kg of 1.25% tribromoethanol into the tail vein. The anesthetized mice were fixed in a clean environment and the abdomen of the mice was disinfected with povidone-iodine.

[0039] (2) Surgical exposure: After holding the mouse skin with autoclaved tissue forceps, make a longitudinal incision of about 1.5-2 cm in length in the lower middle part of the mouse abdomen with sterilized ophthalmic scissors, and cut open the skin and peritoneum in sequence to open the abdominal cavity. Use sterilized blunt forceps to turn the mouse intestine to one side to expose the uterus (containing the embryo and placenta) located in the posterior part of the abdominal cavity.

[0040] (3) Virus injection: Using a 25μL microsyringe equipped with a 34G ultrafine needle, administer a dose of 5×10 12 A 5 μL (vg / mL) solution of PSCAAV-U6-ShRNA (AAV 2 / 9 chimeric serum) was slowly injected into the placental labyrinth region of each placenta. After injection, the mouse uterus and embryo were gently returned to the abdominal cavity.

[0041] (4) Postoperative suturing and care: Using sterilized tissue forceps and needle holders, as well as 4-0 absorbable sutures and curved needles, the peritoneum and skin layers were sutured sequentially using interrupted suturing. After suturing, the abdominal wound was immediately disinfected with povidone-iodine, and then mupirocin ointment (Bactroban) was sprayed on the wound surface to prevent infection.

[0042] (5) Postoperative recovery and observation: After surgery, pregnant mice were placed alone on a 37°C warm water bed or constant temperature mat to maintain their body temperature until they were fully awake from anesthesia. The mice's mental state, activity and wound healing were observed for several days after surgery. If necessary, the wound was disinfected and medication was applied again.

[0043] 4. Sample testing

[0044] Animals were sacrificed on day 5 (E18.5) after injection, and placental tissue was collected. The expression of the viral vector in the placental tissue was observed by immunofluorescence staining, and the infection efficiency between the two groups was compared. The expression levels of the target protein and mRNA in the placental tissue were detected by Western blotting and qPCR, respectively, and the gene knockdown efficiency was quantitatively analyzed.

[0045] Immunofluorescence results as follows Figure 2 As shown, the placental tissue in the placental targeted injection group had a strong viral infection signal, while the tail vein injection group and the negative control group showed almost no infection, indicating that the viral vector can be highly specifically enriched in the targeted injected placenta.

[0046] Western Blot results are as follows: Figure 3 As shown, the target protein bands in the tail vein injection group and the negative control group were similar in intensity to the internal control protein bands, indicating strong signals. This suggests that tail vein injection cannot effectively knock down the expression of the target gene, and the negative control virus itself does not affect gene expression. Figure 3 A). Compared with the tail vein injection group and the negative control group, the target protein band signal was significantly weakened in the placental targeted injection group ( Figure 3 B), the qPCR mRNA level statistical plot also showed that the mRNA expression level in the placental-targeted injection group was significantly lower than that in the negative control group (B), Figure 3 C), indicating that the placental targeted injection method of the present invention can achieve efficient gene knockdown.

[0047] The above results confirm that the method of the present invention can achieve efficient and specific placental tissue gene interference, and avoid systemic off-target infection.

[0048] Example 2: Security and Success Rate Statistics of the Modeling Method

[0049] In continuous application, this surgical method achieved a 100% success rate, indicating that the technical procedure is stable and controllable. Postoperative animal recovery was excellent, with no intraoperative deaths or infections observed in the mouse wounds. The survival rate of pregnant mice exceeded 90%, demonstrating that this invasive procedure has limited impact on the mother and pregnancy process. Furthermore, the effective gene interference success rate of the target placenta exceeded 85%, significantly higher than traditional intravenous injection methods, ensuring the efficiency and consistency of model construction.

[0050] In summary, the placental targeted injection method provided by this invention achieves efficient and specific gene interference while taking into account surgical safety and model stability, and has good reproducibility.

[0051] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for constructing a mouse placental-targeting gene interference model, characterized in that, Includes the following steps: Pregnant mice were anesthetized and underwent abdominal surgery to expose the uterus containing the target placenta; Adeno-associated viruses carrying gene interference elements are directly injected into the target placental tissue; Suture the wound.

2. The method according to claim 1, characterized in that, The steps for exposing the uterus and placenta include: making an incision in the abdomen of the pregnant mouse to open the abdominal cavity, and then dissecting the intestines to expose the uterus and placenta located in the posterior part of the abdominal cavity.

3. The method according to claim 1, characterized in that, The injection is performed via microinjection.

4. The method according to claim 1, characterized in that, The adeno-associated virus serotype is selected from one of AAV2, AAV9, or AAV2 / 9.

5. The method according to claim 1, characterized in that, The gene interference element is a short hairpin RNA.

6. The method according to claim 1, characterized in that, The pregnant mice were in the embryonic stage from day 12.5 to day 15.

5.

7. The method according to claim 1, characterized in that, The injection target site is the labyrinthine region of the placenta.

8. The method according to claim 1, characterized in that, In the injection step, the amount of virus injected per placenta was 1 x 10 10 vg - 5 x 10 10 vg, in a volume of 1-5 μL.

9. A mouse placental-targeting gene interference model constructed by the method of any one of claims 1 to 8.

10. The use of the method according to any one of claims 1 to 8 in the preparation of animal models for studying pregnancy-related diseases.

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