CN117265005A - Method for constructing abortive mouse model and application thereof - Google Patents
Method for constructing abortive mouse model and application thereof Download PDFInfo
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- CN117265005A CN117265005A CN202311208218.1A CN202311208218A CN117265005A CN 117265005 A CN117265005 A CN 117265005A CN 202311208218 A CN202311208218 A CN 202311208218A CN 117265005 A CN117265005 A CN 117265005A
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- C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
Abstract
The invention provides a method for constructing an abortive mouse model, which comprises the step of constructing the abortive mouse model by specifically knocking out Furin genes of mouse endometrial cells. The mouse model constructed by the invention has the phenotypes of embryo implantation failure, embryo absorption, abortion, embryo quantity reduction and the like, accords with the symptoms and pathological processes of recurrent abortion of human beings, and has good operation controllability and good stability. Can be used for researching occurrence and development mechanism and prevention and treatment measures of recurrent abortion and embryo planting failure, and is used for developing and researching new medicines.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a construction method and application of an abortive mouse model.
Background
Embryo implantation is the first critical event in establishing physiological communication between blastocyst stage embryos and the mother's uterus, involving a large number of molecular regulation and signal communication between different cell types. The success or failure of the embryo implantation process determines the normal development trend of the embryo and the final pregnancy outcome. Embryo implantation failure is the primary cause of early pregnancy failure. The embryo transfer failure may be due to poor embryo quality, insufficient endometrial receptivity or an inability of the implanted embryo and the endometrium to interact simultaneously. Two-thirds of the embryo planting failures are caused by insufficient endometrial receptivity due to endometrial stromal cell proliferation and deciduation disorders.
Recurrent abortion (RSA) is a major challenge in reproductive medicine, with complex etiology involving a number of factors including genetic, anatomical, endocrine, immune and thrombogenic factors. RSA is defined as two or more consecutive abortions before 20 weeks of gestation. The etiology of RSA is complex and can be attributed to several factors: genetic factors: chromosomal abnormalities are the most common cause of recurrent abortion, accounting for 50-60% of all cases. These abnormalities may be parental (balanced translocation) or embryonic (aneuploidy); anatomic factors: uterine deformities, intrauterine adhesions, and uterine fibroids can interfere with implantation and placental development, resulting in abortion. Endocrine factors: diseases such as polycystic ovary syndrome (PCOS), thyroid disease, and diabetes can disrupt the hormone balance necessary to maintain pregnancy. Immune factors: certain autoimmune diseases and heterogeneous immune responses can lead to RSA. For example, antiphospholipid syndrome (APS) is an autoimmune disease that increases the risk of thrombosis and pregnancy abortion. Thrombosis factor: susceptible to thrombosis, a condition that increases the risk of thrombosis, can lead to placental insufficiency and RSA.
Because of the medical ethical constraint of human experiments, animal models become important tools for researching the occurrence and the development of abortion and exploring prevention and treatment measures, but no animal model capable of well reflecting the severity of recurrent abortion is available at present. After successful modeling, a critical phenotype of recurrent abortion in humans may occur in part or in whole. The abortion animal model provides guarantee for the characteristics of recurrent abortion diseases, pathogenesis, disease prognosis, research and development of intervention strategies for recurrent abortion, new medicine treatment and the like.
Patent application CN201010217681.9 relates to a method for constructing an animal model, in particular to an animal experimental research model for researching, diagnosing and treating pathogenesis of autoimmune recurrent abortion and antiphospholipid antibody syndrome. The mice model of autoimmune recurrent abortion is established by induction of BALB/c mice by uterine cavity injection of human beta 2 GP-1. The animal model construction method is safe, reliable, simple, convenient and economic, reduces the modeling time of the autoimmune RSA mouse model, improves the modeling success rate and the modeling efficiency, and has good application prospect. But this modeling method is not ideal in terms of safety and modeling efficiency.
Therefore, there is a need in the art for a new method of constructing abortive mouse models and uses thereof.
Disclosure of Invention
The invention firstly provides a method for constructing an abortive mouse model, which comprises the step of constructing the abortive mouse model by specifically knocking out Furin genes of mouse endometrial cells.
In a specific embodiment, the loxP site is inserted into both ends of the Furin gene of a mouse for labeling and effectively knocking out the Furin gene, and the resulting mouse having the loxP site inserted into both ends of the Furin gene is designated as a loxP-flox-Furin mouse, or as Furin f/f A mouse; then mating and backcrossing the loxP-flox-Furin mice with tool mice carrying Pgr-Cre enzyme for purification to obtain the abortive mouse model, namely Furin f/f Pgr-Cre mice, otherwise known as Furin d/d And (3) a mouse.
In a specific embodiment, after obtaining a model of aborted mice, the model mice Furin f/f Female mice of/Pgr-Cre mice and model mice Furin f/f Pgr-Cre miceMale mice were caged to breed more model mice and control mice.
In a specific embodiment, the ratio of male to female cages is 3:1 or greater.
In a specific embodiment, female Furin is initially selected f/f Mice carry the flox sequence, whereas male mice are tool mice carrying the Pgr-Cre enzyme. And initially selecting male Furin f/f Mice carry the flox sequence, while female mice are tool mice carrying the Pgr-Cre enzyme, which can result in higher breeding efficiency in mice.
In a specific embodiment, the mouse is an SPF class C57BL/6 mouse.
The invention also provides application of the abortive mouse model, namely the abortive mouse model prepared by the construction method is used for drug development for treating and/or preventing recurrent abortion or recurrent planting failure of animals and humans.
The present invention also provides a method for screening a drug for treating and/or preventing recurrent abortion or recurrent planting failure in animals and humans, the method comprising the steps of: 1) Applying the drug to be tested to the mouse model constructed by the construction method; 2) And analyzing and evaluating the treatment effect of the drug to be tested, and selecting the test drug capable of obviously improving the pathological characteristics of the model mice.
The invention has at least the following beneficial effects:
1. the invention adopts the mouse model to simulate the natural state of the human body to carry out experiments so as to study pathogenesis and treatment, thereby reducing the necessity of experiment implementation on the human body and avoiding ethical disputes of the human body experiment.
2. The method has the advantages that the mice die in embryo period by knocking out Furin in a whole body, the abortion mouse model is constructed by specifically knocking out Furin genes of endometrial cells, the survival rate of the mice is high, the knocking-out efficiency is high, the phenotype is stable after the successful construction, and the conditions of symptom alleviation or disappearance and the like can not occur along with the increase of time and mating frequency.
3. The pathological manifestations of the flow production model in the invention include embryo planting failure, embryo absorption, abortion, embryo quantity reduction and other phenotypes. The natural disease state of human body can be better simulated, and the natural disease state is better represented.
4. The time for abortion of the mouse model is relatively early, which is beneficial to shortening the period of observation and experiment.
5. The invention can verify and observe gene knockout from multiple aspects of genes, proteins, organs and the like, and the experimental result is true, credible and convincing.
In general, the invention constructs the aborted mouse model by conditional knockout of the endometrial cell Furin gene, and the mouse model has the phenotypes of embryo planting failure, embryo absorption, abortion, embryo quantity reduction and the like, accords with the symptoms and pathological processes of recurrent abortion of human beings, and has good operation controllability and good stability. Can be used for researching occurrence and development mechanism and prevention and treatment measures of recurrent abortion and embryo planting failure, and is used for developing and researching new medicines.
Drawings
FIG. 1 is a schematic flow chart of a method for constructing abortive mouse models.
FIG. 2 is a schematic diagram showing insertion of loxP site at both ends of Furin gene in mice for labeling and efficient knockout of Furin gene.
FIG. 3 is a diagram showing the verification of Furin gene in the constructed mouse model.
FIG. 4 is a Pgr-Cre gene verification diagram of a constructed mouse model.
FIG. 5 is a protein electrophoretogram of endometrial tissue Furin and internal controls of model and control mice.
FIG. 6 is a graph showing the ratio of the amount of protein expressed in endometrial tissue Furin to that of reference mice in both control and model mice.
FIG. 7 is a graph showing the relative expression amounts of mRNA in endometrial tissues of control mice and model mice.
FIG. 8 is a control mouse (i.e., furin) f/f Mice).
FIG. 9 is a model mouse (i.e., furin d/d Mice).
Fig. 10 is a comparison of the data embodiment of fig. 8 and 9.
FIG. 11 is a diagram showing the pathological pattern of the endometrium tissue HE and immunofluorescent staining of mice pregnant for 5.5 days.
Detailed Description
The loxP site and the flox site in the present invention represent the same meaning.
Furin is a protease that is widely distributed in many different types of cells and tissues and performs important biological functions. It is mainly found in the endoplasmic reticulum system of cells (endoplasmic reticulum, ER) and Golgi apparatus (Golgi apparatus). As an important protein post-modification enzyme, the localization of Furin in cells is closely related to the secretory pathway and processing of proteins. Furin is involved in the cleavage and activation of many protein precursors in the endoplasmic reticulum and golgi apparatus. These precursors include a number of important cytokines, growth factors, receptors and enzymes such as TGF-beta, TNF-alpha, angiogenic factors, and the like. Furin activates these molecules by cleaving their precursors.
During embryonic development, new life originates from the binding of sperm and eggs in the oviduct. Fertilized eggs undergo several rounds of mitosis, forming a crowded cell mass called morula (morula). In the advanced morula stage, the embryo enters the uterine cavity and further differentiates into a blastocyst with two distinct cell populations (inner cell mass and trophectoderm cells). At this time, trophoblast cells attach to the endometrium, an implantation process is initiated, and the implanted embryo stimulates the differentiation of the stromal cells of the uterus into decidua cells. As the placenta forms, the mother is better able to deliver nutrition to the fetus to maintain the fetus to continue developing until delivery is initiated. The pregnancy process is extremely complex, whether the embryo can be normally implanted into the endometrium is the key of successful pregnancy, and embryo implantation failure is the main reason for early pregnancy failure.
The FURIN gene (FUR) is located in human chromosome 15 and mouse chromosome 7, and its encoded protein consists of 794 amino acids, including N-terminal signal peptide, prodomain, catalytic domain, P-domain and C-terminal transmembrane domain. The shearing activity was obtained by two self-sheared furins.Furin acting substrates are very wide, such as HIV surface glycoprotein gp160, protgfβ, tgfβ1, BMP family, hepcidin regulatory proteins, etc., and play an important role in tumor invasion, cardiovascular diseases, bacterial virus infection, lipid metabolism, etc. Studies have shown that systemic knockout Furin mice are embryonic lethal and conditional knockout oocytes Furin develop follicular dysplasia and infertility. While conditional knockdown of Furin expression in the placenta will lead to dysgenesis of embryonic development. In particular, mice have been studied for detecting potential physiological functions of a target gene in the uterus of the mice using a progesterone receptor (PGR) driven Cre (PGR-Cre) tool. In order to study Furin function in female reproduction, the learner had Furin flox/flox (Furin fl/fl ) Mice were hybridized with Zp3-Cre mice and Gdf9-Cre mice, respectively, and it was found that knockout of oocyte Furin resulted in specific destruction of oocyte, manifested as follicular dysplasia and infertility. In addition, furin-shRNA packaged lentivirus infected blastocyst-stage (E3.5) embryos, and the blastocysts were implanted into the uterus of pseudopregnant female mice after incubation to obtain a mouse model of placenta site-specific knockout of Furin gene, which was found to lead to embryo development disorder.
Applicant's studies indicate that endometrial knockout Furin eventually results in embryo implantation failure, miscarriage, by interfering with key factors of embryo implantation, the process of deciduation of the endometrial stroma. The obvious activation of the Furin signal pathway in decidua tissues of recurrent abortion patients shows that Furin participates in early embryo implantation process, and the results lay a scientific basis for constructing an abortion animal model for specifically knocking out Furin genes in endometrium.
The invention aims to provide a construction method for constructing an abortive mouse model by specifically knocking out a mouse endometrial cell Furin gene, wherein the abortive mouse model can better reduce the pathological manifestation of the disease, accords with the symptoms and pathological processes of recurrent abortion of human beings, and has good operation controllability and good stability. Can be used for researching occurrence and development mechanism of recurrent abortion and embryo implantation failure, preventing and treating measures and new medicine research.
The invention provides a construction method of abortive mouse model, which inserts loxP sequence into mouse Furin geneIs used for marking and effectively knocking out Furin gene, and the obtained mouse with the loxP site inserted into the two ends of Furin gene is named as loxP-flox-Furin mouse or Furin f/f And (3) a mouse. Then mating and backcrossing the loxP-flox-Furin mice with tool mice carrying Pgr-Cre enzyme for purification to obtain the abortive mouse model, namely Furin f/f Pgr-Cre mice. The genotype of a 3-week-old mouse is typically determined by examining its toe or tail. Further, by collecting Furin f/f The expression level of Furin protein and mRNA in endometrial tissue of Pgr-Cre mice is used for verifying the knockout of Furin genes in endometrial cells of the mice and further determining whether an aborted mouse model is successfully constructed. At the same time, furin f/f Female mice of Pgr-Cre mice and Furin f/f Male mice of Pgr-Cre mice were caged to breed more model mice. The method comprises the following steps: male and female mice of model mice were selected for mating with female mice for breeding more model mice and control mice. Offspring mice can be genotyped about 3 weeks after birth, whether the Furin gene is wild type (no flox sequence is inserted into both DNA strands in a pair), heterozygous (only one strand of both DNA strands in a pair has flox sequences inserted at both ends of its Furin gene), or f/f type (both DNA strands in a pair have flox sequences inserted at both ends of its Furin gene), and f/f type mice are selected for analysis of their Pgr-Cre genotypes to identify whether model mice or control mice. The Furin gene of the control mice was f/f type and the Pgr-cre genotype was negative, so that the Furin gene was not knocked out in such mice. The mating success rate can be guaranteed to the greatest extent by controlling the proportion of the male cage to the female cage to be 1:3, so that the orderly performance of a model building experiment is guaranteed, and the problem that the experiment period is prolonged due to long-time mating failure in the experimental process is avoided.
In general, the invention provides a method for constructing an abortive mouse model, which constructs the abortive mouse model by specifically knocking out endometrial cell furin genes, wherein the mouse model has characteristic symptoms of embryo planting failure, embryo absorption, abortion and embryo quantity reduction, accords with symptoms and pathological processes of recurrent abortion of human beings, and has good operation controllability and good stability. Can be used for researching occurrence and development mechanisms and prevention and treatment measures of recurrent abortion and repeated planting failure, and is used for developing and researching new medicines.
In the present invention, we insert loxP site into both ends of the Furin gene of mouse to label and knock out the Furin gene effectively, and the obtained mouse with loxP site inserted into both ends of Furin gene is named as loxP-flox-Furin mouse or Furin f/f And (3) a mouse. Then mating and backcrossing the loxP-flox-Furin mice with tool mice carrying Pgr-Cre enzyme for purification to obtain the abortive mouse model, namely Furin f/f PGR-Cre mice. The genotype of a 3-week-old mouse is typically determined by examining its toe or tail. Further, by collecting Furin f/f The expression level of Furin protein and mRNA in endometrial tissue of Pgr-Cre mice is used for verifying the knockout of Furin genes in endometrial cells of the mice and further determining whether an aborted mouse model is successfully constructed. In the uterus, progesterone receptors are mainly distributed in the endometrium by construction of Furin f /f The Pgr-Cre mouse model can specifically knock out the Furin gene in endometrial cells, and further provides a reliable model for elucidating the action mechanism of the Furin gene in embryo implantation.
The present invention will be further described with reference to the accompanying drawings and the following examples, but the scope of the present invention is not limited to the following examples.
Example 1
FIGS. 1 and 2 are flowcharts showing the construction of a mouse model for endometrial cell-specific Furin gene knockout in the present invention. Selecting SPF-class C57BL/6 background mice, inserting loxP sequences at two ends of Furin gene, integrating target gene with loxP fragment into original genome, and using to mark and knock out Furin gene so as to obtain loxP-flox-Furin mice, namely Furin f/f And (3) a mouse. Then mating and backcrossing the loxP-flox-Furin mice with tool mice carrying Pgr-Cre enzyme for purification to obtain a mouse model of the endometrium cell specific knockout Furin gene, namely Furin f/f PGR-Cre mice.
FIGS. 3 and 4 are graphs of genetic verification of the constructed mouse model. FIG. 3 is a diagram showing the verification of Furin gene, and FIG. 4 is a diagram showing the verification of Furin genePgr-Cre gene verification map. FIG. 3 shows the verification of the genotype of the mouse Furin gene by nucleic acid gel electrophoresis, wherein the leftmost column is a standard reagent bright band for indicating the size of nucleic acid, and the 2 nd to 4 th columns are heterozygous, namely, the two ends of the Furin gene with one nucleic acid chain in the paired DNA chains are inserted with loxP sequences, the nucleic acid size is 332bp, the two ends of the Furin gene with the other nucleic acid chain are not inserted with loxP sequences, and the nucleic acid size is 271bp; columns 5-10 are homozygous f/f, i.e. the two DNA strands have loxP sites inserted at both ends of the Furin gene, and the nucleic acid sizes of both strands are 332bp. Selecting the Furin gene homozygote type, and verifying the genotype of the PGR-Cre gene by nucleic acid electrophoresis, wherein the verification result is shown in figure 4. In FIG. 4, the leftmost column shows the bright bands of standard reagents, which are used to indicate the size of nucleic acids. Wherein, the number 4-5 from the left is PGR-Cre genotype positive abortive mouse model, the nucleic acid size is 661bp, and the mice corresponding to the rest multiple bands (namely, the number 2, 3, 6 and 7) are all PGR-Cre genotype negative. These Furin genes are homozygous (i.e., f/f type) and PGR-Cre genotype negative mice (Furin genes on endometrial cells are not knocked out) can be used as control mice for the mouse model of the invention. As can be seen from FIGS. 3 and 4, the mouse model Furin obtained by the present invention d/d The genotype of the mice is homozygous for the Furin gene and carries the PGR-Cre gene.
FIG. 5 shows the protein electrophoresis of endometrial tissue Furin and reference in model and control mice, wherein the first row is the protein of mice homozygous for the Furin gene (i.e., f/f type) and having a molecular weight of about 87kDa, and the second row is a schematic representation of GAPDH, an reference protein, and having a molecular weight of about 36kDa. Control mice (i.e., furin) with homozygous Furin gene (i.e., f/f type) and negative PGR-Cre genotype in the left three bands of the first row f/f Mice), the expression level of the protein is higher as can be seen from the protein electrophoresis diagram of the mice); while the right three bands in the first row are all homozygous (i.e., f/f type) Furin gene and the PGR-Cre genotype is positive (i.e., furin) d/d Mice), the expression level of Furin protein in the endometrial tissue of the mice is reduced or even eliminated.
FIG. 6 is a graph showing the ratio of the amount of protein expressed by endometrial tissue Furin to that of reference in control mice and model mice, and FIG. 6 is a data representation of FIG. 5, wherein each data point represents one mouse. As can be seen from FIG. 6, the endometrial tissue of the control mice showed significantly higher protein expression than that of the model mice, which were statistically different, and p <0.05 (p < 0.05).
FIG. 7 is a graph showing the relative expression amounts of mRNA in endometrial tissues of control mice and model mice. As can be seen from FIG. 7, the relative expression of mRNA from Furin endometrial tissue of control mice was significantly higher than that of model mice, with statistical differences, and p <0.001 (p < 0.001).
As can be seen from FIGS. 5 to 7, western blotting and mRNA expression levels showed that the conditional knockout mice, i.e., furin, compared with the control mice d/d The expression level of Furin in mouse endometrial tissue is significantly reduced at the protein level and the mRNA level.
Figures 8-10 are general diagrams of the reproductive system of mice pregnant for 5.5 days and corresponding data graphs. Wherein FIG. 8 is a control mouse (i.e., furin) f/f Mice), FIG. 9 is a model mouse (i.e., furin) d/d Mice). Two mice are included in both figures 8 and 9. Fig. 10 is a representation of the data of fig. 8 and 9, with each data point representing a mouse. As shown in FIGS. 8 to 10, furin was compared with the control mice d/d Mice showed a significant decrease in the number of embryo implantation points, and a phenotypic change in embryo uptake and abortion.
FIG. 11 is a diagram showing the pathological pattern of the endometrium tissue HE and immunofluorescent staining of mice pregnant for 5.5 days. Wherein the upper row is a control mouse (i.e., furin f/f Mouse) endometrial tissue HE and immunofluorescent staining pathology, and the lower row is model mice (i.e., furin) d/d Mice) endometrial tissue HE and immunofluorescent staining pathology. The first column is HE staining, the second column is magnification 100 times, the third column is magnification 200 times, and the magnification of the objective lens is respectively 10 times and 20 times. The second and third columns are each Furin immunofluorescent staining pathology plots (Furin, 1:500, abcam, abc3460). As shown in fig. 11, with Furin f/f Furin compared to mice d/d Mouse FThe urin expression is obviously reduced.
The foregoing is a further detailed description of the invention in connection with specific preferred embodiments, and is not intended to limit the practice of the invention to such description. It will be apparent to those skilled in the art that several simple deductions and substitutions can be made without departing from the spirit of the invention, and these are considered to be within the scope of the invention.
Claims (8)
1. A method for constructing a model of aborted mice, the method comprising constructing a model of aborted mice by specifically knocking out Furin genes of mouse endometrial cells.
2. The method for constructing a model of aborted mice according to claim 1, wherein the insertion of loxP sites at both ends of the Furin gene of the mice is used for labeling and efficient knockout of Furin gene, and the resulting mice having the insertion of loxP sites at both ends of Furin gene are designated as loxP-flox-Furin mice, or as Furin f/f A mouse; then mating and backcrossing the loxP-flox-Furin mice with tool mice carrying Pgr-Cre enzyme for purification to obtain the abortive mouse model, namely Furin f/f Pgr-Cre mice, otherwise known as Furin d/d And (3) a mouse.
3. The method for constructing an abortive mouse model according to claim 2, wherein after obtaining the abortive mouse model, the model mouse Furin f/f Female mice of/Pgr-Cre mice and model mice Furin f/f Male mice of Pgr-Cre mice were caged to breed more model mice and control mice.
4. The method for constructing an abortive mouse model according to claim 1, wherein the ratio of male to female cages is 3:1 or more.
5. The method for constructing a model of abortive mice according to any one of claims 1 to 4, wherein the model is selected at the beginningFemale-selecting Furin f/f Mice carry the flox sequence, whereas male mice are tool mice carrying the Pgr-Cre enzyme.
6. The method for constructing a model of aborted mice according to any one of claims 1 to 4, wherein the mice are SPF class C57BL/6 mice.
7. An application of an abortive mouse model, which is characterized in that the abortive mouse model prepared by the construction method of any one of claims 1-6 is used for drug development for treating and/or preventing recurrent abortion or recurrent planting failure of animals and humans.
8. A method of screening for a drug for the treatment and/or prevention of recurrent abortion or recurrent planting failure in animals and humans, said method comprising the steps of:
1) Applying a drug to be tested to a mouse model constructed by the construction method according to any one of claims 1 to 6;
2) And analyzing and evaluating the treatment effect of the drug to be tested, and selecting the test drug capable of obviously improving the pathological characteristics of the model mice.
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| KEMAL OZBILGIN: "Distribution of furin, TNF-α, and TGF-β2 in the endometrium of missed abortion and voluntary first trimester termination cases", 《ANAL QUANT CYTOPATHOL HISTPATHOL》, vol. 37, no. 2, 30 April 2015 (2015-04-30) * |
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