Male testicular dysplasia mouse model as well as construction method and application thereof

Abstract

The invention provides a male testicular dysplasia mouse model as well as a construction method and application thereof, and belongs to the field of animal model construction. The construction method comprises the following steps: S1, inserting two loxP sites in the upstream and downstream of a mouse Dhx37 gene, and simultaneously expressing Cre recombinase to produce an F0-generation heterozygote mouse; s2, the F0-generation heterozygote mice and the wild mice are mutually mated to generate F1-generation heterozygote mice; then, the F1-generation heterozygote mice are mutually mated to generate F2-generation homozygote mice; s3, the F2 generation homozygote mouse and the whole body / tissue specific tool mouse are mated with each other to generate an F3 generation heterozygote Amh-Cre mouse; and then the F3 generation heterozygote Amh-Cre mice are mated with each other to generate an F4 generation homozygote Amh-Cre mouse. Male testis dysplasia mice can be massively produced through gene knockout and amplification breeding, and the stable character can be used as a basis for testis development research and drug screening.

Description

Technical Field

[0001] The present invention relates to the field of animal model construction, and particularly to a male mouse model with testicular dysplasia, a construction method thereof, and an application thereof. Background Art

[0002] Infertility is a common disease that seriously affects human health, and its incidence has been increasing year by year in recent years. Infertility refers to a situation where a couple has normal sexual life and has not been pregnant for one year or more without taking any contraceptive measures. Male infertility is the main or partial cause of 20 - 70% of infertile couples. The causes of male infertility are extremely complex, involving the anatomical structure and function of the reproductive system, infection, endocrine, genetic factors, etc.

[0003] However, the current lack of establishment of testicular development models limits the research on male infertility and the exploration of drugs. Therefore, there is an urgent need for a male mouse model with testicular dysplasia to provide a theoretical basis. Summary of the Invention

[0004] The purpose of the present invention is to provide a male mouse model with testicular dysplasia, a construction method thereof, and an application thereof. The present invention can produce a large number of male mice with testicular dysplasia through gene knockout and amplification reproduction, and the traits are stable and can be used as the basis for testicular development research and drug screening.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a construction method of a male mouse model with testicular dysplasia, including the following steps:

[0007] S1. By inserting two loxP sites into the mouse Dhx37 gene and simultaneously expressing Cre recombinase, the target gene knockout is completed to produce Dhx37 gene knockout mice;

[0008] The Dhx37 gene knockout mice are F0 generation heterozygous mice Dhx37 Flox / + ;

[0009] S2. The F0 generation heterozygous mice Dhx37 Flox / + are crossbred with wild mice to produce F1 generation heterozygous mice Dhx37 Flox / + ; Then the F1 generation heterozygous mice Dhx37 Flox / + are crossbred with each other to produce F2 generation homozygous mice Dhx37 Flox / Flox ;

[0010] S3. The F2 generation homozygous mice Dhx37 Flox / Flox are crossbred with a whole body / tissue-specific tool mouse to produce F3 generation heterozygous Amh-Cre mice Dhx37Flox / + ; Then, the F3 heterozygous Amh-Cre mice Dhx37 Flox / + were interbred with each other to produce the F4 homozygous Amh-Cre mice Dhx37 Flox / Flox .

[0011] Preferably, the specific method in S1 is: LoxP sites are inserted upstream and downstream of the exon 2 region of the Dhx37 gene, and after mating with Amh-cre mice, mice with targeted knockout of the Dhx37 gene in the testicular tissue at embryonic day 12.5 are obtained.

[0012] Preferably, the wild mice in S2 are mice with the same background as Dhx37 Flox / + .

[0013] Preferably, the F4 homozygous Amh-Cre mice Dhx37 Flox / Flox in S3 show that: the testicular volume and the ratio of testicular weight to body weight are significantly smaller than those of wild-type mice, the serum testosterone level is significantly lower than that of wild-type mice, the number of spermatogonia is reduced, the number of sperm in the lumen is reduced, the number of testicular Sertoli cells is reduced, and vacuolization appears in the testicular tissue, indicating successful modeling.

[0014] The present invention also provides a male mouse model with testicular dysplasia, which is constructed by the above construction method.

[0015] The present invention also provides the application of the male mouse model with testicular dysplasia in testicular development research.

[0016] The present invention also provides the application of the male mouse model with testicular dysplasia in screening drugs for promoting testicular development.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention can produce a large number of male mice with testicular dysplasia through gene knockout and amplification reproduction, and the traits are stable, which can be used as the basis for testicular development research and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a comparison diagram of the testicular volume of mice;

[0020] Figure 2 It is a diagram of the ratio of testicular weight to mouse body weight;

[0021] Figure 3 It is a diagram of serum testosterone level;

[0022] Figure 4 It is an H&E staining diagram of testicular sections of DHX37 gene-deficient group and wild-type mice;

[0023] Figure 5Electron micrograph of sperm count in the cauda epididymis under a microscope;

[0024] Figure 6 Quantification result graph of sperm count in the testis and epididymis of mice;

[0025] Figure 7 Dhx37 - / - Graph showing a decrease in the number of germ cells in the group;

[0026] Figure 8 Graph showing a decrease in the number of Sertoli cells in the testicular tissue of DHX37 gene - deficient mice;

[0027] Figure 9 Graph showing an increase in the number of TUNEL - positive cells in the testicular tissue of DHX37 gene - deficient mice. Detailed implementation manners

[0028] Now, the various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0031] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are only exemplary.

[0032] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open - ended terms, meaning including but not limited to.

[0033] In the present invention, unless otherwise specified, "room temperature" and "normal temperature" are both calculated as 25 ± 2 °C.

[0034] Unless otherwise specified, the raw materials used in the following examples of the present invention are all commercially available.

[0035] Example 1

[0036] DHX37 Conditional Knockout Mouse Breeding Strategy

[0037] 1. Gene knockout principle:

[0038] In the Cre-loxP knockout technology, Cre recombinase recognizes the loxP site. Based on the relative position and orientation of the loxP sites, recombination can lead to gene deletion, insertion, inversion, or translocation.

[0039] By inserting two loxP sites upstream and downstream of the mouse Dhx37 gene and simultaneously expressing Cre recombinase, the target gene knockout is finally completed to produce knockout mice. loxP is a 34-base pair DNA sequence for Cre recombinase recognition and binding. Flox represents the state where a gene / gene fragment is flanked by two loxP sites, and this gene or fragment can be recombinantly operated by Cre recombinase.

[0040] 2. Mouse source: Mice carrying the Flox-Dhx37 allele were purchased from Cyagen Biosciences (Suzhou) Inc.; 1 line of Amh-Cre mice.

[0041] 3. Obtaining Dhx37 Gene-targeted Homozygous Knockout Mice (Dhx37-CKO) through Breeding

[0042] Breeding process: (1) Expanding the population: Heterozygous F0 mice (Dhx37 Flox / + ) were mated with wild mice to produce F1 heterozygotes; (2) Obtaining homozygotes: F1 heterozygous mice (Dhx37 Flox / + ) were mated with each other to produce F2 homozygotes (Dhx37 Flox / Flox ); (3) Obtaining heterozygous knockout mice: F2 homozygous mice (Dhx37 Flox / Flox ) were mated with a whole body / tissue-specific tool mouse to obtain F3 (Dhx37 Flox / + , Amh-Cre); (4) Obtaining homozygous knockout mice: F3 (Dhx37 Flox / + , Amh-Cre) were mated with each other to produce F4 (Dhx37 Flox / Flox , Amh-Cre), that is, CKO homozygotes (Dhx37 - / - ). (Note: Dhx37 - / - refers to homozygous mice, Dhx37 + / -heterozygous mice).

[0043] Example 2

[0044] Identify the testicular development-related indicators of the conditional knockout mice obtained in Example 1

[0045] (1) Testicular volume and the ratio of testis weight to body weight, see Figures 1 - 2 .

[0046] The testicular volume and the ratio of testis weight to body weight of DHX37 gene-deficient mice are significantly smaller than those of wild-type mice

[0047] (2) Comparison of serum testosterone levels, see Figure 3 .

[0048] The serum testosterone level of DHX37 gene-deficient mice is significantly lower than that of wild-type mice.

[0049] (3) Observe testicular tissue by HE staining, see Figure 4 .

[0050] In the Dhx37 gene-deficient mouse group, the number of spermatogonia is reduced, the number of sperm in the lumen is reduced, the number of Sertoli cells in the testis is reduced, and vacuolization appears in the testicular tissue.

[0051] (4) Count the sperm in the cauda epididymis, see Figures 5 - 6 .

[0052] Counting the sperm in the cauda epididymis, microscopic observation and quantification technical results show that the sperm count of DHX37 gene-deficient mice is reduced.

[0053] (5) Reflect the number of germ cells in testicular tissue by immunofluorescence staining of TRA98 expression, see Figure 7 .

[0054] Compared with wild-type mice, the TRA98-positive germ cells in the testicular tissue of Dhx37-deficient mice are significantly reduced.

[0055] A. There are no germ cells in the testis of Dhx37 gene-deficient mice. Immunofluorescence staining of DHX37 and TRA98 (germ cells) in testicular sections of WT and Dhx37- / - mice at P 60. DNA was stained with DAPI. Scale bars represent 100 μm and 50 μm respectively. B. Bar graph shows a decrease in the total number of germ cells in Dhx37- / -.

[0056] (6) Reflect the number of Sertoli cells in testicular tissue by immunofluorescence staining of SOX9 expression, see Figure 8 .

[0057] The number of Sertoli cells in the testicular tissue of DHX37 gene-deficient mice is significantly reduced.

[0058] A, Immunofluorescence staining, with SOX9 labeling Sertoli cells; B, The bar graph of the quantification results is shown.

[0059] (7) The apoptosis of Sertoli cells in testicular tissues was detected by TUNEL staining and SOX9 immunofluorescence labeling, as shown in Figure 9 .

[0060] The apoptosis of Sertoli cells in testicular tissues of DHX37 gene-deficient mice was significantly more than that of wild-type mice. A. Increased apoptosis of cells in testicular tissues of DHX37 gene-deficient mice, immunofluorescence staining of TUNEL and SOX9 (Sertoli cells). DNA was stained with DAPI. The scale bars represent 100 μm and 50 μm respectively. B. The bar graph of the quantification results.

[0061] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing a male mouse model with testicular dysplasia, characterized in that, Including the following steps: S1. By inserting two loxP sites into the mouse Dhx37 gene and simultaneously expressing Cre recombinase, the target gene knockout is completed to produce Dhx37 gene knockout mice; The Dhx37 gene knockout mouse is an F0 generation heterozygous mouse Dhx37 Flox / + ; S2. Mate the F0 generation heterozygous mice Dhx37 Flox / + with wild mice to produce F1 generation heterozygous mice Dhx37 Flox / + ; Then mate the F1 generation heterozygous mice Dhx37 Flox / + with each other to produce F2 generation homozygous mice Dhx37 Flox / Flox ; S3. Mate the F2 generation homozygous mice Dhx37 Flox / Flox with whole-body / tissue-specific tool mice to generate F3 generation heterozygous Amh-Cre mice Dhx37 Flox / + ; then mate the F3 generation heterozygous Amh-Cre mice Dhx37 Flox / + with each other to generate F4 generation homozygous Amh-Cre mice Dhx37 Flox / Flox .

2. The construction method according to claim 1, characterized in that, The specific method in S1 is: inserting LoxP sites upstream and downstream of the exon 2 region of the Dhx37 gene, and mating with Amh-cre mice to obtain Dhx37 gene-targeted knockout mice in testicular tissue at embryonic day 12.

5.

3. The construction method according to claim 1, characterized in that, The wild mice in S2 are mice with the same genetic background as Dhx37 Flox / + mice.

4. The construction method according to claim 1, characterized in that, The F4 generation homozygous Amh-Cre mice Dhx37Flox / Flox in S3 show that: the testicular volume and the ratio of testicular weight to body weight are significantly smaller than those of wild-type mice, the serum testosterone level is significantly lower than that of wild-type mice, the number of spermatogonia is reduced, the number of sperm in the lumen is reduced, the number of testicular Sertoli cells is reduced, and vacuolization appears in the testicular tissue, which is regarded as successful modeling.

5. A male mouse model with testicular dysplasia, characterized in that, Constructed by the construction method described in any one of claims 1 to 4.

6. Application of the male testicular dysplasia mouse model described in claim 5 in testicular development research.

7. Application of the male testicular dysplasia mouse model described in claim 5 in screening drugs for promoting testicular development.

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