A method for constructing a spontaneous premature birth mouse model

Abstract

The present invention provides a method for constructing a spontaneous premature birth mouse model, which belongs to the field of biomedicine. The present invention specifically knocks out the CBS gene of mouse myeloid cells to construct a spontaneous premature birth mouse model, which is mainly manifested by shortened gestational duration and reduced fetal weight, which is consistent with the symptoms and pathological process of spontaneous premature birth in humans, and the model has good operability and high stability, and can be used to study the occurrence and development mechanism and prevention and treatment measures of spontaneous premature birth, as well as for the development and research of new drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for constructing a spontaneous premature birth mouse model. Background Art

[0002] Premature birth is an abnormal initiation of labor, which is clinically defined as birth at 28 weeks of gestation but less than 37 weeks. The cause of premature birth is complex, the mechanism is unclear, and there is no effective prevention and treatment. Most (60%) premature births are natural, caused by premature rupture of membranes (25%) or spontaneous premature birth (35%). The remaining 40% of premature births occur due to medical indications such as multiple pregnancies, fetal malformations, and preeclampsia or chorioamnionitis. At present, due to the unknown cause, there is no effective model to simulate spontaneous premature birth and conduct research. Therefore, the construction of a spontaneous premature birth mouse model can provide a very good research tool for the occurrence, development and treatment of spontaneous premature birth, and provide a basis for drug development.

[0003] Due to the medical ethics restrictions on human experiments, animal models have become an important tool for studying the occurrence and development of spontaneous preterm birth and exploring preventive measures. However, there is currently no animal model of spontaneous preterm birth with a stable phenotype and available for mass breeding. Therefore, there is a need in the art for a method for constructing an animal model of spontaneous preterm birth. Summary of the invention

[0004] The initiation of labor is dynamically and precisely regulated by the immune system, in which innate immune cells, especially myeloid cells, play a very important role in this process. Cystathionine beta synthase CBS is a core member of the transsulfurization pathway, encoded by the CBS gene located on human chromosome 21 (mouse chromosome 17); it is closely related to H2S production and DNA methylation, and is also involved in redox balance, post-transcriptional modification, and metabolic reprogramming in the body. We used Cre-loxp technology to specifically knock out the CBS gene in mouse myeloid cells and verified its knockout, obtaining CBS f / f / LYZ2-Cre and observed its phenotype to obtain a spontaneous premature birth mouse model.

[0005] The present invention first provides a method for constructing a spontaneous premature birth mouse model, which comprises constructing a spontaneous premature birth mouse model by specifically knocking out the CBS gene of mouse myeloid cells.

[0006] In a specific embodiment, loxP sequences are inserted into both ends of the mouse CBS gene to mark the CBS gene. The resulting mice with loxP sequences inserted into both ends of the CBS gene are named loxP-flox-CBS mice, or CBS f / f Mice; then, CBS f / fThe mice were mated with tool mice carrying the LYZ2-Cre enzyme and then f / f The spontaneous premature birth mouse model, namely CBS, was obtained by backcrossing and purification of mice. f / f / LYZ2-Cre mice; the LYZ2-Cre enzyme is myeloid cell-driven Cre.

[0007] In a specific embodiment, after obtaining the spontaneous premature birth mouse model, the model mouse CBS f / f Male CBS / LYZ2-Cre mice and control mice f / f The female mice were caged together to breed more model mice and control mice. f / f Male CBS / LYZ2-Cre mice and control mice f / f The female mice were caged together to breed more model mice and control mice. Half of their offspring were model mice and the other half were control mice. Specifically, about a quarter of the offspring were model mice CBS. f / f / LYZ2-Cre female mice, about a quarter of the control mice CBS f / f Female mice.

[0008] In a specific embodiment, the male-female cage ratio is 3:1 or more.

[0009] In a specific embodiment, the mouse is an SPF grade C57BL / 6 mouse.

[0010] The present invention also provides an application of a spontaneous premature birth mouse model, including the spontaneous premature birth mouse model prepared by the above construction method for use in drug development for treating and / or preventing spontaneous premature birth in animals and humans.

[0011] The present invention also provides a method for screening drugs for treating and / or preventing spontaneous premature birth in animals and humans, comprising the following steps: 1) administering the drug to be tested to the mouse model constructed by the above-mentioned construction method; 2) analyzing and evaluating the therapeutic effect of the drug to be tested, and selecting the test drug that can significantly improve the pathological characteristics of the model mice.

[0012] In general, the present invention provides a method for constructing a premature mouse model, wherein loxP sequences are inserted into both ends of the mouse CBS gene to mark the CBS gene, and the resulting mice are named loxP-flox-CBS mice, or CBS f / f Afterwards, CBS f / f The mice were mated with tool mice carrying LYZ2-Cre enzyme and backcrossed to purify the premature mouse model, namely CBS. f / f / LYZ2-Cre mice. The genotype of the mice was determined by testing the DNA of the tail or toes of the 3-week-old mice. f / f The CBS protein and mRNA expression levels in bone marrow macrophages of LYZ2-Cre mice were analyzed to verify the knockout of CBS in bone marrow macrophages and to further confirm the successful construction of the mouse model.

[0013] The present invention has at least the following beneficial effects:

[0014] 1. The present invention simulates the symptoms and pathological process of spontaneous premature birth in humans through a mouse model, which can provide a very good model for disease research and effectively avoid ethical disputes in human experiments.

[0015] 2. The present invention has strong operability and high repeatability, and the phenotypic observation period is greatly shortened compared with human body research, which is conducive to the development of basic research.

[0016] 3. Since the cause of spontaneous premature birth is unknown, there has been no effective research model before. The invention can provide a certain direction for the study of the cause and mechanism of spontaneous premature birth, as well as prevention and treatment.

[0017] 4. The mouse model of the present invention has an obvious phenotype of spontaneous premature birth, which can fill the gap of the current lack of relevant animal models and is conducive to the observation and design of relevant experiments and the development of relevant drugs.

[0018] 5. The present invention can verify and observe gene knockout from multiple angles such as genes, proteins, tissues and organs, and the experimental results are real, credible and convincing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of knocking out the CBS gene using Cre-loxp technology.

[0020] Figure 2 To build CBS f / f Flowchart of LYZ2-Cre / LYZ2-Cre mice.

[0021] Figure 3 This is the nucleic acid electrophoresis verification of the LYZ2-Cre gene.

[0022] Figure 4 To verify the CBS gene by nucleic acid electrophoresis.

[0023] Figure 5 CBS f / f Mice and mouse models CBS f / f Changes in CBS RNA levels in bone marrow macrophages of / LYZ2-Cre mice.

[0024] Figure 6are the gestation durations of control mice and model mice, respectively, i.e., the time from the appearance of follicles in female mice to the delivery of the first mouse.

[0025] Figure 7 are the numbers of pregnant litters of each control mouse and model mouse, respectively.

[0026] Figure 8 The number of mice with pregnancy loss, that is, the number of absorption points, is the number of each control mouse and model mouse respectively.

[0027] Fig. 9 are the weights of each fetus of control mice and model mice respectively.

[0028] Fig.10 The corresponding placenta weight of each fetus of control mice and model mice respectively.

[0029] Fig.11 This is a picture of the basic morphology of mice during childbirth. DETAILED DESCRIPTION

[0030] Figure 1 and Figure 2 It's CBS f / f / Technical principle and flow chart of LYZ2-Cre mouse construction. SPF wild-type mice were selected, loxP sequences were first inserted at both ends of the CBS gene, and the target gene with loxP fragments was integrated into the original genome to mark and knock out the CBS gene, thereby obtaining loxP-flox-CBS mice, namely CBS f / f Then, loxP-flox-CBS mice were mated with tool mice carrying LYZ2-Cre enzyme and backcrossed to purify, thus obtaining a mouse model with myeloid cell-specific knockout of CBS gene, namely CBS f / f / LYZ2-Cre mice.

[0031] In the present invention, the CBS is customized f / f The mouse process is generally completed by a genetic company. f / f The preparation method of mice is a common technology available to gene companies. Then, a tool mouse of LYZ2-Cre enzyme is constructed. This transgenic mouse can express Cre recombinase in myeloid cells. Since the tool mouse of LYZ2-Cre enzyme is available for direct purchase, it is a mature commodity, so the construction method of the mouse is not described here. In the present invention, CBS is generally used. f / f Mice were used as control mice instead of wild mice because CBS f / f There was no change in the characteristics of the mice.

[0032] Figure 2 In the control mouse CBS customized by Gene Companyf / f The CBS gene of mice is homozygous with loxP sequences inserted in both nucleotides. The commercialized tool mice carrying LYZ2-Cre enzyme purchased from a gene company have both nucleotides of the Cre gene positive and homozygous; their offspring mice CBS f / - The CBS gene of / LYZ2-Cre mice is a heterozygote with only one nucleotide inserted into the loxP sequence, and its Cre gene is also a heterozygote with only one nucleotide positive. However, the Cre enzyme only needs one nucleotide positive in the Cre gene to be effective, which means that the heterozygote of the Cre gene is as effective as the homozygote. f / - / LYZ2-Cre mice were then compared with control mice CBS f / f Mice were backcrossed; half of the resulting offspring mice were homozygous with loxP sequences inserted into both nucleotides of the CBS gene, and the other half were heterozygous with only one nucleotide of the CBS gene inserted into the loxP sequence. The CBS heterozygous mice were discarded, and half of the CBS homozygous mice were Cre enzyme positive, i.e., model mice CBS f / f / LYZ2-Cre mice, the other half of the CBS homozygotes were Cre enzyme negative, that is, the control mice CBS f / f mice. Therefore, in general, CBS f / - CBS / LYZ2-Cre mice and control mice f / f Among the offspring mice obtained after backcrossing, 1 / 4 are model mice, which is the total number of female and male model mice. Therefore, the probability of female and male model mice is 1 / 8 each.

[0033] Those skilled in the art will know that if the CBS gene of mice is completely knocked out, it will be lethal. In the present invention, the CBS gene of mouse myeloid cells is conditionally knocked out. The myeloid cells include monocytes, mature macrophages and neutrophils. In the present invention, the LYZ2 is a tool enzyme representing myeloid cells.

[0034] Figure 3 and Figure 4 It is to verify whether the mouse is successfully constructed at the DNA level. Figure 3 For LYZ2-Cre gene verification, Figure 3 The leftmost column is DNA maker, which is used to indicate the molecular weight of DNA, and the first column from the left is a blank control, which has no reagent added, the second column WT is the tail DNA of wild-type mice, which do not carry the cre gene, and the third and fourth columns Cre are the tail DNA of two LYZ2-Cre tool mice and the model mice prepared by the present invention, and the nucleic acid size is 700bp. The mice corresponding to the remaining multiple bands are all LYZ2-Cre genotype negative. Figure 4For CBS gene verification, Figure 4 The 1st to 3rd columns are wild-type mice WT, whose band is 355bp, and the 4th to 7th columns are four control mice CBS f / f Mice or model mice, the band is 437bp. Figure 4 The rightmost column is DNA maker, which is used to indicate the molecular weight of DNA. Figure 3 Only LYZ2-Cre enzyme is verified to be positive. Figure 4 In this test, we only verify whether the mouse CBS gene is positive. A positive CBS gene indicates that the loxp sequence has been successfully inserted, and a double positive CBS gene and LYZ2-Cre enzyme indicates that the CBS gene has been successfully knocked out in the mouse myeloid cells. Figure 4 Among the four mice marked with CBS, the second one from the left is a model mouse that is double positive for the CBS gene and LYZ2-Cre enzyme, so it shows the cut CBS fragment at 200bp, while the other three mice are control mice that are positive for the CBS gene and negative for the LYZ2-Cre enzyme.

[0035] Figure 5 CBS f / f Mice and mouse models CBS f The changes in CBS RNA levels in bone marrow macrophages of / f / LYZ2-Cre mice verified that the CBS gene of the model mice was specifically knocked out at the transcriptional level. Figure 5 As can be seen, the relative expression level of CBS mRNA in myeloid cells of control mice was significantly higher than that of model mice, and there was a statistical difference between the two, and p<0.01 (** represents p<0.01). Figure 5 The BMDM in the figure refers to bone marrow macrophages, which represent myeloid cells. The CBSFLox in the figure refers to the CBS of the control mouse. f / f Mouse, the LYZ CBS in the picture is the model mouse CBS f / f / LYZ2-Cre mice. The present invention also conducted a large number of animal experiments to observe whether the spontaneous premature birth mouse model constructed by the present invention is stable, its operability and repeatability. First, we used the Cre-loxp technology to specifically knock out the myeloid CBS gene, and obtained CBS through corresponding breeding and purification. f / f / LYZ2-Cre mice, and the knockout was verified by DNA electrophoresis and QPCR. f / f / LYZ2-Cre male mice were mated with female mice, and their pregnancy phenotypes were observed at the time of delivery. Through a large number of repeated experiments, we found that compared with the control group, there was no significant difference in the size, litter size, and abortion rate of the knockout mice. However, the gestation period of the knockout mice was significantly shortened, and the fetal weight was reduced, which was consistent with the phenotypic characteristics of spontaneous premature birth.

[0036] Male mice and female mice were caged together in a ratio of 1:2. The cages were put together at 8 o'clock in the evening of the first day, and the plugs were checked at 8 o'clock in the morning of the second day. If a plug was found, it was recorded as 0.5 days. Figure 6 It is the time from the appearance of the plug to the delivery of the first mouse. Figure 7 is the number of litters per mouse, Figure 8 is the number of mice with pregnancy loss, Fig. 9 and Fig.10 is the weight of each fetus and its corresponding placenta weight, Fig.11 This is a picture of the basic morphology of mice during childbirth.

[0037] Figure 6 In this study, we first obtained female and male model mice that were both positive for the CBS gene and the LYZ2-Cre enzyme, and allowed them to mate to produce offspring. We also obtained control mice CBS f / f The female and male mice were allowed to mate and produce offspring. The gestation period of wild-type mice WT was 19.5 days, and the 6 control mice CBS f / f The mice were basically the same as wild-type mice, and they gave birth naturally on day 19.3, while the six model mice gave birth on an average of 18.75 days of gestation. There was a statistical difference between the two, and p<0.0001 (*** represents p<0.0001).

[0038] Figure 6 to Figure 10 The left column represents the model mice with double positive CBS gene and LYZ2-Cre enzyme, that is, the female model mice mate with the male model mice, and the right column represents the control mouse CBS f / f Mice, namely control female mice were mated with control male mice. Figure 8 The absorption points in the figure represent the number of mice with abortion. ns in the figure represents that there is no statistical difference between the model mice and the control mice. Fig. 9 It can be seen that the average litter weight of the model mice is about 1g, while the control mice CBS f / f The average litter weight of mice was about 1.2g. There was a statistical difference between the two, and p<0.0001 (*** represents p<0.0001). Fig.10 It can be seen that there is no statistical difference in the placenta weight of the litters of model mice and the placenta weight of the litters of control mice. Fig.10 and Fig.11 In mice, there is one placenta for each litter. Fig.11 In the figure, the rightmost litter of mice is a naturally born litter of mice, and its placenta has been eaten by the mother mouse. After the first litter of mice is born, the inventor will kill the mother mouse and take out the remaining litters of mice and their placentas.

[0039] In general, the present invention provides a method for constructing a spontaneous premature birth mouse model by specifically knocking out the CBS gene of mouse myeloid cells. The premature birth mouse model can better restore the pathological manifestations of the disease, is consistent with the symptoms and pathological process of spontaneous premature birth in humans, and has good controllability and stability. It can be used to study the occurrence and development mechanism of spontaneous premature birth, preventive and therapeutic measures, and for new drug research.

[0040] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions and substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope of the present invention.

Claims

1. A method for constructing a spontaneous premature birth mouse model, characterized in that: The method comprises constructing a spontaneous premature birth mouse model by specifically knocking out the CBS gene of mouse myeloid cells.

2. The method for constructing a spontaneous premature birth mouse model according to claim 1, characterized in that: The loxP sequence was inserted into both ends of the mouse CBS gene to mark the CBS gene. The resulting mice with loxP sequences inserted into both ends of the CBS gene were named loxP-flox-CBS mice, or CBS f / f Mice; then, CBS f / f Mice were mated with tool mice carrying the LYZ2-Cre enzyme and CBS f / f The spontaneous premature birth mouse model, namely CBS, was obtained by backcrossing and purification of mice. f / f / LYZ2-Cre mice; the LYZ2-Cre enzyme is myeloid cell-driven Cre.

3. The method for constructing a spontaneous premature birth mouse model according to claim 2, characterized in that: After obtaining the spontaneous premature birth mouse model, the model mouse CBS f / f Male CBS / LYZ2-Cre mice and control mice f / f Female mice were caged together to breed more model mice and control mice.

4. The method for constructing a spontaneous premature birth mouse model according to claim 3, characterized in that: The ratio of male to female in the cage should be 3:1 or above.

5. The method for constructing a spontaneous premature birth mouse model according to any one of claims 1 to 4, characterized in that: The mice are SPF grade C57BL / 6 mice.

6. Use of a spontaneous premature birth mouse model, characterized in that: The spontaneous premature birth mouse model prepared by the construction method described in any one of claims 1 to 5 is used for the development of drugs for treating and / or preventing spontaneous premature birth in animals and humans.

7. A method for screening drugs for treating and / or preventing spontaneous premature birth in animals and humans, characterized in that: The method comprises the following steps: 1) administering the drug to be tested to the mouse model constructed by the construction method according to any one of claims 1 to 5; 2) Analyze and evaluate the therapeutic effects of the drugs to be tested, and select the test drugs that can significantly improve the pathological characteristics of the model mice.

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