Construction method of vagina-free uterus fallopian tube mouse model

Abstract

The invention provides a construction method of a non-vaginal uterus fallopian tube mouse model, and belongs to the technical field of biology. The invention provides a construction method of a vagina-free uterus fallopian tube mouse model, which comprises the following steps: mating a Wt1 + / -mouse with an Amhr2-Cre tool mouse to obtain Wt1 + / -; the Amhr2-Cre mouse and the flox mouse with the Wt1 gene being conditionally knocked out copulate, the Wt1 gene in the mouse muller tube interstitial cell genome can be specifically knocked out, and the vagina-free uterus fallopian tube mouse model which is stable in phenotype and high in exogenesis rate is obtained. The method disclosed by the invention provides an important bridge for research and development of medicines for treating female genital malformation and treatment of clinical diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for constructing a mouse model of absence of vagina, uterus and fallopian tubes. Background Art

[0002] Female genital tract malformation refers to the changes in the fusion, canalization and development of the primordial internal genitalia and the evolution of the external genitalia during the differentiation process of female reproductive organs, resulting in various developmental abnormalities. The most severe form of female genital tract malformation is congenital absence of uterus and vagina syndrome (MRKH syndrome).

[0003] Establishing a genital tract malformation model is helpful for better studying the pathogenesis, mechanism of MRKH syndrome and screening of related drugs. The unique comprehensive advantages of gene-edited mouse models in model organisms are not only manifested in the similarity between genetics and physiology and humans, but also in the favorable factors such as low cost, rapid and convenient breeding, and clear genetic background. By constructing a mouse disease model, it is helpful to deeply reveal the mechanism of action of potential pathogenic genes, provide tools for preclinical evaluation research of drugs, and become an important bridge for the effective transformation from basic research on drug development to clinical disease treatment application. However, the existing female genital tract malformation mice have problems of unstable phenotypes and low penetrance, which seriously hinder the progress of the treatment of genital tract malformation diseases. Summary of the Invention

[0004] In view of this, the present invention provides a method for constructing a mouse model of absence of vagina, uterus and fallopian tubes, and by specifically knocking out the Wt1 gene in mouse Müllerian duct interstitial cells, a female genital tract malformation mouse model with stable phenotype and high penetrance can be obtained.

[0005] To achieve the above object, the present invention provides the following technical solutions: The present invention provides a method for constructing a mouse model of absence of vagina, uterus and fallopian tubes, comprising the following steps: Mating male Wt1 + / - mice with female Amhr2-Cre tool mice to obtain Wt1 + / - ; Amhr2-Cre mice; Mating female Wt1 + / - ; Amhr2-Cre mice with male Wt1 gene flox conditionally knocked out mice to obtain a mouse model of absence of vagina, uterus and fallopian tubes.

[0006] Preferably, the reagents for detecting the Wt1 + / - mice include Wt1+ / - Primer set; The Wt1 + / - The primer set includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:2.

[0007] Preferably, the reagent for detecting the Wt1 + / - ; Amhr2-Cre mouse includes the Wt1 + / - primer set and Amhr2- Cre primer set; The Amhr2-Cre primer set includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:4.

[0008] Preferably, the Wt1 gene flox mouse is obtained by Rosa flox / flox mouse mating with Wt1 flox / flox mouse.

[0009] Preferably, the reagent for detecting the Wt1 gene flox mouse includes Wt1 flox / flox primer set and Rosa flox / flox primer set; The Wt1 flox / flox primer set includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:5 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:6; The Rosa flox / flox primer set includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:7 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:8.

[0010] Preferably, the reagent for detecting the mouse model without vagina, uterus and oviduct includes the Wt1 flox / flox primer set, the Rosa flox / flox primer set, Wt1 + / - primer set and Amhr2-Cre primer set; The Wt1 + / -The primer set includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:2; The Amhr2-Cre primer set includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:4.

[0011] Preferably, in the vagina-uterus-oviduct absent mouse model, the absence of the uterus is manifested as at least one of the following: absence of uterine lumen, absence of myometrium, and absence of endometrium.

[0012] Preferably, the absence of endometrium includes the non-expression of the endometrial gland marker FOXA1.

[0013] Preferably, the ovaries of the vagina-uterus-oviduct absent mouse model develop normally.

[0014] Preferably, the normal ovarian development is manifested as at least one of the following: normal primordial follicles, normal primary follicles, normal secondary follicles, and normal corpus luteum.

[0015] The present invention has the following advantages compared with the prior art: The present invention provides a method for constructing a vagina-uterus-oviduct absent mouse model. A male Wt1 + / - mouse is mated with a female Amhr2-Cre tool mouse to obtain Wt1 + / - ; Amhr2-Cre mice; the female Wt1 + / - ; Amhr2-Cre mice are mated with male conditionally knockout Wt1 gene flox mice to obtain a vagina-uterus-oviduct absent mouse model. The method of the present invention knocks out the Wt1 gene in the genome of mouse Müllerian duct stromal cells, and a vagina-uterus-oviduct absent mouse model with stable phenotype can be obtained. Examples of the present invention compared the reproductive tract development of knockout mice and control mice. The results showed that compared with control mice, the knockout mice had no vaginal opening, the uterus did not develop, there was a thin layer of myometrium, and no obvious oviduct structure was seen. In addition, the method provided by the present invention does not affect the body size of knockout mice, nor does it affect the development of their ovaries and follicles at all levels, providing an important bridge for the drug research and development for the treatment of female reproductive tract malformations and the treatment of clinical diseases. Description of the Drawings

[0016] Figure 1Morphological comparison diagrams of the external genitalia and uterus of knockout mice and control mice at adulthood (8w); among them, A: comparison result of mouse body size; B: comparison result of absence of vaginal opening in mice; C: morphology of reproductive organs of control mice; D: morphology of reproductive organs of knockout mice; E: H&E staining result of uterine cross-section of control mice; F: H&E staining result of cord-like structure of knockout mice; Figure 2 Morphological diagrams of ovaries (3w) of knockout mice and control mice; among them, A and C are ovaries of control mice; B and D are ovaries of knockout mice; Figure 3 Expression result diagrams of endometrial glands and myometrium markers of knockout mice and control mice; among them, A: expression result of uterine ACTA of control mice; B: expression result of uterine ACTA of knockout mice; C: expression result of uterine FOXA1 of control mice; D: expression result of uterine FOXA1 of knockout mice; Figure 4 Color development diagrams of the urogenital system of E12.5-day mouse embryos under a fluorescence microscope; among them, A and B are images of control mice, C and D are images of knockout mice, and O represents the ovary and M represents the mesonephros in A - D; Figure 5 Wholemount staining diagram of the Mullerian duct of E13.5-day embryonic mice; A: wholemount staining result of the mesonephros of control mice, B and C: Wt1 Wholemount staining result of the mesonephros of knockout mice, with PAX8 labeling the Mullerian duct epithelium and GFP labeling the Mullerian duct interstitial cells. Detailed implementation methods

[0017] The present invention provides a method for constructing a mouse model without vagina, uterus and fallopian tubes, comprising the following steps: Male Wt1 + / - Mice are mated with female Amhr2-Cre Tool mice to obtain Wt1 + / - ; Amhr2-Cre Mice; Female Wt1 + / - ; Amhr2-Cre Mice are mated with conditional knockout male Wt1 Genes of flox Mice to obtain a mouse model without vagina, uterus and fallopian tubes.

[0018] In the present invention, the preparation method of the Wt1 + / - Mice preferably utilizes the principle of gene recombination, and the replacement vector pWT and Wt1The gene has a homeodomain with a length of 10 kb. The first exon of the gene and the sequence 0.5 kb upstream thereof are replaced by the homeodomain in the replacement vector pWT; Wt1 The gene is used for positive selection, and the thymidine kinase ( PGK-neo-poly(A) ), gene is used for negative selection. The replacement vector pWT is introduced into the J1 embryonic stem cell line. After 8 days of screening, clones resistant to G418 and FIAU are screened. The screened clones are injected into C57BL / 6 blastocysts, and the resulting chimeric mice are crossed with wild-type C57BL / 6 mice, and the mutation is transmitted through the germ line. thymidine kinase, TK The specific preparation method of the mice refers to the prior art (Kreidberg JA, Sariola H, Loring JM, Wt1 + / - WT-1 is required for early kidney development. Cell. 1993;74:679-91.). In the method of the present invention, introducing et al. mice can improve the knockout efficiency, avoid the problem of incomplete knockout, and at the same time improve the stability of the phenotype of the knockout mice, thereby obtaining a mouse model without vagina, uterus and oviduct. Wt1 + / - In the present invention, the reagent for detecting the

[0019] mice preferably includes Wt1 + / - a primer set; the Wt1 + / - primer set includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:2. Since Wt1 + / - homozygous mice cannot survive, using the genome of the mouse to be tested as a template and amplifying with the Wt1 - / - primer set, when an amplification product of 250 bp is obtained, the mouse to be tested is Wt1 + / - heterozygous, and when no amplification product of 250 bp is obtained, the mouse to be tested is Wt1 + / - homozygous. Wt1 + / + After obtaining the

[0020] mice, in the present invention, the male Wt1 + / - mice are mated with female Wt1 + / - tool mice to obtain Amhr2-Cre mice. Wt1 + / - ; Amhr2-Cre mice.

[0021] In the present invention, Amhr2-Cre The method for preparing the tool mouse is preferably to use the gene targeting method to introduce a Cre DNA fragment of -neomycin (neo) sequence into mouse embryonic stem cells, and this fragment integrates with the mouse chromosome through the principle of homologous recombination. Cre-neo The sequence is inserted into the Amhr2 5th exon of the Amhr2-Cre For the specific method for preparing the tool mouse, it is preferably to refer to the prior art (Jamin SP, Arango NA, Mishina Y, Hanks MC, Behringer RR. Requirement of Bmpr1a for Mullerian duct regression during male sexual development. Nat Genet. 2002;32:408-10.).

[0022] In the present invention, male Wt1 + / - mice are mated with female Amhr2-Cre tool mice, and Wt1 + / - ; Amhr2-Cre mice are obtained through genotype identification and screening. The reagents for detecting the Wt1 + / - ; Amhr2-Cre mice include Amhr2-Cre a primer set and the Wt1 + / - primer set; the Amhr2-Cre primer set includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:4. Using the genome of the mouse to be tested as a template, amplification is performed with the Wt1 + / - primer set to obtain an amplification product of 250 bp, and amplification is performed with the Amhr2-Cre primer set to obtain an amplification product of 550 bp. The mouse to be tested is Wt1 + / - ; Amhr2-Cre mice.

[0023] After obtaining Wt1 + / - ; Amhr2-Cre mice, the present invention preferably mates female Wt1 + / - ; Amhr2-Cre mice with male conditionally knockout Wt1 gene floxMice were mated to obtain a mouse model without vagina, uterus and oviduct.

[0024] In the present invention, the conditional knockout Wt1 gene flox mice ( Wt1 flox / flox ; Rosa flox / flox ) are preferably obtained by mating Rosa flox / flox mice with Wt1 flox / flox mice. Specifically, Rosa flox / flox mice are mated with Wt1 flox / flox mice, and the mice are screened by genotype identification to obtain Wt1 + / flox ; Rosa + / flox mice; Wt1 + / flox ; Rosa + / flox mice are mated with Wt1 + / flox ; Rosa + / flox mice, and the mice with conditional knockout of Wt1 gene flox mice ( Wt1 flox / flox ; Rosa flox / flox ) are obtained by genotype identification and screening. The Rosa flox / flox mouse is a dual-fluorescent Cre reporter mouse, which expresses membrane-targeted Tomato (mT) before Cre -mediated LoxP excision and expresses membrane-targeted green fluorescent protein (mG) after excision. When there is no Cre enzyme, the pCA promoter (chicken β-actin core promoter with a CMV enhancer) drives the expression of membrane-targeted Tomato, which terminates at the first pA sequence (terminator); when there is Cre enzyme, after Cre -mediated recombination, the mT + pA sequence is excised, enabling the pCA promoter to drive the expression of green fluorescent protein (GFP). Rosa flox / floxThe specific preparation method of the mouse preferably refers to the prior art (Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent Cre reporter mouse. Genesis. 2007;45:593-605.). Wt1 flox / flox The preparation method of the mouse is preferably to insert one Wt1 on both sides of the 8th and 9th exons of the gene LoxP site. The 8th and 9th exons encode Wt1 the 2nd and 3rd zinc finger binding domains of the Wt1 protein, and the latter two are Wt1 flox / flox The specific preparation method of the mouse refers to the prior art (Gao F, Maiti S, Alam N, et al. The Wilms tumor gene, Wt1, is required for Sox9 expression and maintenance of tubular architecture in the developing testis. Proc Natl Acad Sci U S A. 2006;103:11987-92.).

[0025] In the present invention, the reagent for detecting the Wt1 gene flox in the Wt1 flox / flox mouse preferably includes Rosa flox / flox a primer set and Wt1 flox / flox a primer set; the Rosa flox / flox primer set includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:5 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:6; the Wt1 flox / flox primer set includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:7 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:8. Using the genome of the mouse to be tested as a template and amplifying with the Wt1 + / + primer set, only one amplification product of 150 bp is obtained, indicating that the mouse to be tested is Wt1flox / flox Homozygous, and obtaining an amplification product of 150 bp and an amplification product of 250 bp simultaneously, indicating that the mouse to be tested is Wt1 + / flox heterozygous. Using the genome of the mouse to be tested as a template and amplifying with the Rosa flox / flox primer set, only obtaining an amplification product of 250 bp, indicating that the mouse to be tested is Rosa flox / flox homozygous, only obtaining an amplification product of 330 bp, indicating that the mouse to be tested is Rosa + / + homozygous, obtaining an amplification product of 250 bp and an amplification product of 330 bp simultaneously, indicating that the mouse to be tested is Rosa + / flox heterozygous. Using the genome of the mouse to be tested as a template and amplifying with the Wt1 flox / flox primer set, only obtaining an amplification product of 250 bp, and using the Rosa flox / flox primer set to amplify, only obtaining an amplification product of 330 bp, the mouse to be tested is a Wt1 conditionally knockout flox gene mouse ( Wt1 flox / flox ； Rosa flox / flox )). In the present invention, the reagent for detecting the mouse model without vagina, uterus and oviduct ( Wt1 - / flox ; Rosa + / flox ； Amhr2-Cre ) preferably includes the Wt1 flox / flox primer set, the Rosa flox / flox primer set, the Wt1 + / - primer set and the Amhr2-Cre primer set. Using the genome of the mouse to be tested as a template and amplifying with the Wt1 flox / flox primer set, the Rosa flox / flox primer set, the Wt1 + / - primer set and the Amhr2-Cre primer set respectively, obtaining amplification products of 250 bp, 250 bp, 250 bp and 550 bp, indicating that the mouse to be tested is Wt1 - / flox ; Rosa + / flox ; Amhr2-Cre.

[0026] In the present invention, theWt1 + / - Primer set, Amhr2-Cre Primer set, Wt1 flox / flox Primer set and Rosa flox / flox The reaction system of PCR for the primer set is preferably independently 10 μL of 2× Taq DNA polymerase, 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of template, and 7 μL of double-distilled water (ddH2O). The reaction program of PCR is preferably: pre-denaturation: 94 °C for 2 min; denaturation: 94 °C for 30 s, annealing: 63 °C for 30 s, extension: 72 °C for 45 s, 35 cycles; extension: 72 °C for 10 min; storage: 4 °C.

[0027] In the present invention, the mice used are of C57BL / 6J background. The age of the female or male mice used for mating is preferably 8 - 10 w, more preferably 9 w. The number ratio of female mice to male mice is (1 - 3):1, more preferably 2:1. The time for co-housing the mice is preferably in the afternoon, more preferably from 16:00 to 18:00 in the afternoon. Defining the genetic background of the mating mice, as well as the gender and age of mating, is beneficial to obtaining a mouse model of vaginal, uterine, and fallopian tube absence with stable phenotype and high penetrance. The method of the present invention has been verified by repeated experiments, and the obtained mouse model of vaginal, uterine, and fallopian tube absence has a stable phenotype and high penetrance.

[0028] In the present invention, in the mouse model of vaginal, uterine, and fallopian tube absence constructed by the above method, the absence of the uterus preferably mainly shows at least one of the following: absence of uterine lumen, absence of uterine muscle layer, and absence of endometrium. The absence of endometrium preferably includes non-expression of the endometrial gland marker FOXA1. The ovaries of the mouse model are preferably normally developed. The normal development of the ovaries preferably shows at least one of the following: normal primordial follicles, normal primary follicles, normal secondary follicles, and normal corpus luteum.

[0029] The method of the present invention can specifically knockout the Wt1Genes were used to obtain a mouse model of vagina-free, uterus-free, and oviduct-free with stable phenotypes and high penetrance. In the examples of the present invention, the reproductive tract development of knockout mice and control mice was compared. The results showed that, compared with the control mice, the knockout mice had no vaginal opening, the uterus did not develop, presenting a thin cord-like structure, without the uterine lumen, uterine epithelium, endometrium, and uterine myometrium, and no obvious oviduct structure was observed. There was no obvious abnormality in body size, and there was no obvious abnormality in primordial follicles, primary follicles, secondary follicles, and corpora lutea. The uteruses of mice at 5w, 3w, and 1d after birth were similar in phenotype to those of 8w knockout mice, indicating that the uterus of knockout mice did not develop from birth, and the development of the ovaries and follicles at all levels of knockout mice was not affected. Microscopic observation of knockout mouse embryos showed that the Mullerian ducts of knockout mouse embryos were dysplastic. Whole-mount staining was performed on mouse embryos throughout the embryonic period, and the results showed that the Mullerian ducts of the knockout mice could not extend.

[0030] To further illustrate the present invention, a method for constructing a mouse model of vagina-free, uterus-free, and oviduct-free provided by the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0031] 1. Experimental animals The mice used in the present invention are all of C57BL / 6J background and are housed in the SPF-class animal house of the Experimental Animal Center of the Institute of Zoology, Chinese Academy of Sciences. The breeding of experimental animals follows the "Regulations on the Administration of Experimental Animals" in Beijing and the regulations of animal health institutions. The breeding environment is 12h of light and 12h of darkness alternating every day, the temperature is maintained at 24±1°C, and the humidity is kept at about 35±4%. Animal experiments are carried out strictly in accordance with the regulations of the Animal Ethics Committee.

[0032] (1) Amhr2-Cre The preparation method of the tool mice refers to the prior art (Jamin SP, Arango NA, Mishina Y, Hanks MC, Behringer RR. Requirement of Bmpr1a for Mullerian duct regression during male sexual development. Nat Genet. 2002;32:408-10.).

[0033] (2) Rosa flox / floxThe method for preparing mice refers to the prior art Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent Cre reporter mouse. Genesis. 2007;45:593-605.

[0034] (3) Wt1 flox / flox Mice: Using the principle of gene recombination in mouse embryonic cells Wt1 Insert one on each side of exons 8 and 9 of the gene LoxP site. Exons 8 and 9 encode Wt1 The second and third zinc finger binding domains of the protein, and the latter two are Wt1 The key structural domains for the molecule to exert its biological function. Wt1 flox / flox The specific method for preparing mice refers to the prior art (Gao F, Maiti S, Alam N, et al. The Wilms tumor gene, Wt1, is required for Sox9 expression and maintenance of tubular architecture in the developing testis. Proc Natl Acad Sci U S A. 2006;103:11987-92.).

[0035] (4) Wt1 + / - Mice: By constructing a replacement vector pWT, which has a 10 kb homologous domain with the Wt1 gene; Using the principle of gene recombination to replace the first exon of the Wt1 gene and the 0.5 kb sequence upstream of it, the PGK-neo-poly(A) gene is used for positive selection, while the thymidine kinase ( thymidine kinase, TK ) gene is used for negative selection. Introduce pWT into the Jl embryonic stem cell line (embryonic stem cells), and after 8 days of screening, clones resistant to G418 and FIAU are screened. Inject the screened clones into C57BL / 6 blastocysts, and the resulting chimeric mice are crossed with wild-type C57BL / 6 mice, and the mutation is transmitted through the germ line. Wt1 + / - The specific method for preparing mice refers to the prior art (Kreidberg JA, Sariola H, Loring JM, et al.WT-1 is required for early kidney development. Cell.1993;74:679-91.)

[0036] 2. The experimental instruments and their corresponding manufacturers are shown in Table 1.

[0037] Table 1 Experimental Instruments

[0038] 3. Reagent Information (1) The experimental reagents and their corresponding reagent companies are shown in Table 2.

[0039] Table 2 Experimental Reagents

[0040] (2) Preparation of Commonly Used Experimental Reagents Preparation of 4% PFA-PBS buffer (1000 ml): Weigh 40 g of paraformaldehyde (PFA) and place it in a beaker. Add 500 - 800 ml of 1×PBS, heat to about 60°C, and continuously stir (or use magnetic stirring) until the powder is completely dissolved. Usually, a little 1N NaOH needs to be added dropwise to make the solution clear. Finally, make up to 1000 ml with 0.01M PBS and mix well. Adjust the pH to 7.4. After preparation, it can be aliquoted and stored at -20°C.

[0041] (3) The antibodies, their usage concentrations, and the corresponding reagent companies are shown in Table 3.

[0042] Table 3 Antibody Information

[0043] Example 1 Preparation Method of a Mouse Model without Vagina, Uterus, and Oviduct 1. Mouse Reproduction Strategy (1) Mate male Wt1 + / - mice with female Amhr2-Cre tool mice, and screen the obtained Wt1 + / - ; Amhr2-Cre mice through genotype identification; (2) Mate Rosa flox / flox mice with Wt1 flox / flox mice, and screen the obtained Wt1 + / flox ; Rosa + / flox mice; Mate Wt1 + / flox; Rosa + / flox Mice were mated with Wt1 + / flox ; Rosa + / flox mice, and conditional knockout Wt1 gene flox mice ( Wt1 flox / flox ; Rosa flox / flox ) were obtained by genotype identification and screening; (3) Female Wt1 + / - ; Amhr2-Cre mice were mated with male Wt1 flox / flox ; Rosa flox / flox mice, and knockout mice with the Wt1 gene knocked out in mouse Müllerian stromal cells ( Wt1 - / flox ; Rosa + / flox ; Amhr2- Cre ) were used as a mouse model without vagina, uterus and fallopian tubes; knockout mice ( Wt1 + / flox ; Rosa + / flox ; Amhr2-Cre ) obtained by genotype identification were used as control mice.

[0044] When breeding mice, introducing Wt1 + / - can avoid the problem of incomplete knockout caused by low efficiency of Cre mice. For the enzyme, it is only necessary to knockout two Flox sites on one sister chromatid. Introducing Cre mice can label Rosa flox / flox positive cells. Cre

[0045] 2. Methods for preparing mating mice and checking vaginal plugs On the afternoon of the previous day at 16-18 o'clock, fertile (9-week-old) female and male mice were caged together. The ratio of female to male mice was 2:1. Sufficient mouse food and water were provided, and the numbers, genotypes, genders, dates of birth and dates of caging of the mice were marked in detail on the breeding card.

[0046] On the morning of the second day between 8 and 10 o'clock, vaginal plugs were checked. That is, the vaginal orifice of the female mouse was gently opened with forceps to observe whether there was a milky coagulum. If there was a vaginal plug, it indicated that the female and male mice had mated, which was recorded as E0.5; if there was no vaginal plug, the female and male mice were separated and caged, and mating was continued on the afternoon of the same day, and vaginal plugs were checked the next day.

[0047] 3. Mouse labeling​ Two weeks after birth, mice can be ear-tagged and marked. Gently hold the back and neck skin of the mouse with the left hand, fix it in the palm, and clamp the mouse's tail with the little finger and ring finger of the left hand. Arrange the ear tags in order, hold the ear tag pliers with the right hand, gently clamp an ear tag, quickly clamp the ear tag on the right ear of the mouse, and check whether the ear tag is clamped tightly. Gently cut off about 5 mm of the end of the mouse's tail with ophthalmic scissors and place it in a 1.5 ml EP tube, and mark the corresponding ear number on the tube. For mice within two weeks after birth, if genotype identification is urgently needed, the method of cutting toes is used for marking.

[0048] 4. Genotype identification of mice Put the tail or toes of the mouse to be identified into a 1.5 mL EP tube, add 99 μL of lysis buffer and 1 μL of 20 mg / mL proteinase K, place it on a shaker, and lyse at 56 °C and 270 rpm for at least 4 h. Then, inactivate proteinase K using a 95 °C metal bath for later use, or store it temporarily in a 4 °C refrigerator.

[0049] Use the above lysis product as a template for polymerase chain reaction (PCR) to perform genotype identification.

[0050] The primer sequences, annealing temperatures, and corresponding band sizes for genotype identification of mice are shown in Table 4.

[0051] Table 4 Primer sequences, annealing temperatures, and band sizes for genotype identification of mice

[0052] Note: F: forward primer; R: reverse primer; R1: reverse primer for wild-type strand; R2: reverse primer for strand containing LoxP site.

[0053] The PCR reaction system for genotype identification of mice is as follows: 2×Taq DNA polymerase: 10 μL, forward primer: 1 μL, reverse primer: 1 μL, template: 1 μL, double-distilled water (ddH2O): 7 μL.

[0054] The PCR reaction procedure is as follows: Pre-denaturation: 94 °C for 2 min; denaturation: 94 °C for 30 s, annealing: 63 °C for 30 s, extension: 72 °C for 45 s, 35 cycles; extension: 72 °C for 10 min; storage: 4 °C.

[0055] Gel preparation: Prepare a 2% agarose gel with 1×TAE solution.

[0056] Electrophoretic imaging: Use 1×TAE solution as the electrophoresis buffer; load the samples in sequence, add 10 μL of PCR product to each well, and add 6 μL of Marker to at least one well in each row. If the band is within 1000 bp, select 100 bp plus marker; if the band is above 1000 bp, select 2000 bp plus marker. Apply a voltage of 100 v and run electrophoresis for 30 min, then image under a gel imager.

[0057] Example 2 Mouse genital tract detection 1. Mouse genital tract tissue collection Euthanize the mice by cervical dislocation. Place the knockout mice and their littermate control mice side by side and take pictures (mainly of the perineum) for record. Cut open the lower abdomen, enter the abdominal cavity, bluntly separate the tissues around the uterus with fine forceps, and free the uterus, fallopian tubes, and ovaries. Bluntly separate the ligaments and tissues around the cervix downward, remove the bladder, cut open the symphysis pubis to expose the vagina. Bluntly separate the tissues around the vagina until the vaginal orifice. Cut the skin around the vaginal orifice along the vaginal orifice.

[0058] Place the isolated female mouse reproductive systems in a 10 cm petri dish containing PBS solution, and remove the fat around the organs with fine forceps under a stereomicroscope. Place the reproductive systems of the knockout mice and their littermate control mice side by side and take pictures for record.

[0059] 2. Tissue fixation Fix with 4% PFA phosphate buffer solution overnight. Wash 3 times with 1×PBS, and then place in 70% alcohol. If not embedded immediately, it can be temporarily stored in a 4℃ refrigerator after being placed in 70% alcohol.

[0060] 3. Paraffin embedding 1) Place in embedding cassette: Put the fixed tissue into the embedding cassette, and mark relevant information on the embedding cassette, including the genotype, age, sex, tissue name, tissue number, and embedding time of the source animal.

[0061] 2) Gradient alcohol dehydration: Immerse the embedding cassette in 70%, 80%, and 90% alcohol at room temperature for 1 h each, and gently shake on a shaker during the immersion process to fully dehydrate the tissue. Then place it in 100% alcohol at room temperature for 40 min × 2 times.

[0062] 3) Clearing: Place the embedding cassette in a solution of xylene: absolute ethanol (1:1, v / v) at room temperature for 30 min, and then place it in xylene at room temperature for 30 min.

[0063] 4) Wax infiltration: Place the paraffin in the oven the day before the experiment and melt the wax at 60℃ to ensure sufficient wax liquid on the day of the experiment. Place the embedding cassette in 70℃ paraffin I, paraffin II, and paraffin III for 1 h each in sequence.

[0064] 5) Embedding: Turn on the wax embedding box 3 hours before embedding to melt the wax. Drop a small amount of wax into the embedding tray first, place the tissue in it and position it in the center, cover it with a white plastic fixing box, and then drop a small amount of wax. Then place it on an ice table and wait for it to solidify. The solidified tissue is stored in a 4°C refrigerator and awaits sectioning.

[0065] 4. Hematoxylin-eosin staining (HE staining) 1) Deparaffinization: Sequentially place the paraffin sections in xylene 1, xylene 2, and xylene 3 in a fume hood and soak them for 10 minutes each; 2) Rehydration with gradient alcohol: Sequentially soak the sections in 100%, 100%, 100%, 95%, 80%, and 70% alcohol for 5 minutes each; 3) Washing: Wash with PBS 3 times, 5 minutes each time; 4) Hematoxylin staining: Drop hematoxylin staining solution on the glass section and stain for 45 seconds, and terminate the staining with tap water; 5) Differentiation: Place the stained section in 0.1% hydrochloric acid alcohol for 3 - 5 seconds for differentiation; 6) Blueing: Place the differentiated section in tap water and rinse the section gently with running water for 10 - 30 minutes to make it blue; 7) Eosin staining: Prepare the eosin staining solution as a working solution by mixing it with 95% alcohol at a ratio of 1:1, stain for 30 seconds (the time can be adjusted according to the staining situation at any time); after staining, wash with tap water for 5 minutes, observe at any time, and then place it in 95% alcohol; 8) Dehydration and clearing: Sequentially place the stained section in 95%, 100%, and 100% alcohol in a fume hood and soak for 30 seconds each, then place the section in xylene 1, xylene 2, and xylene 3 and soak for 3 minutes each; 9) Sealing: Take out the section from xylene 3, quickly drop 1 - 2 drops of neutral balsam, cover it with a coverslip, and avoid forming air bubbles. Air dry it in a fume hood, and take a photo after the section is dry.

[0066] 5. Immunofluorescence staining of paraffin sections 1) Steps 1 - 3 refer to the HE staining of paraffin sections; 2) Antigen retrieval: Place the section in a citrate antigen retrieval solution and boil it in a microwave oven at high power (about 5 minutes); immediately switch to low power after boiling and continue to boil for 15 minutes; cool it naturally to room temperature; 3) PBS washing: Wash the section with 1×PBS 3 times, 5 minutes each time; 4) Blocking: The blocking solution used for immunofluorescence staining is 5% bovine serum albumin (BSA) diluted with 0.3% TritonX-100; Use a histochemical pen to circle the tissue on the glass slide, add 200 μL of the blocking solution, and block at room temperature for 1 h; 5) Primary antibody incubation: Dilute the blocking solution with 0.3% TritonX-100 to 1% BSA, and the latter is used as the primary antibody diluent. Dilute the antibody according to the antibody concentration specified in the instruction manual. Pour off the blocking solution on the slide, shake off as much as possible, add 200 μL of the primary antibody diluent, and block at room temperature for 1 h; 6) PBS washing: Wash the slide with 1×PBS 3 times, 5 min each time; 7) Secondary antibody incubation: Dilute the fluorescent secondary antibody with 1×PBS according to the instruction manual, add DAPI, and incubate at room temperature in the dark for 1 h; 8) PBS washing: Wash the slide with 1×PBS 3 times, 5 min each time in the dark; 9) Mounting: Take out the slide from PBS, quickly drop 1 - 2 drops of anti-fluorescence quenching agent, cover with a cover slip, and avoid forming air bubbles. Place the slide in a dark box and store it at 4℃ temporarily, and observe under a fluorescence microscope as soon as possible.

[0067] 5. Immunohistochemical staining of paraffin sections 1) Steps 1 - 4 refer to the immunofluorescence staining of paraffin sections; 2) Blocking: The blocking solution used for immunohistochemical staining is 5% BSA diluted with 1×PBS. Use a histochemical pen to circle the tissue on the glass slide, add 200 μL of the blocking solution, and block at room temperature for 1 h; 3) Primary antibody incubation: Dilute the blocking solution with 1×PBS to 1% BSA, and the latter is used as the primary antibody diluent. Dilute the antibody according to the antibody concentration specified in the instruction manual. Pour off the blocking solution on the slide, shake off as much as possible, add 200 μL of the primary antibody diluent, and block at room temperature for 1 h; 4) PBS washing: Wash the slide with 1×PBS 3 times, 5 min each time; 5) Remove peroxidase in the tissue: Immerse the slide in 3% H2O2 in the dark for 10 min; 6) PBS washing: Wash the slide with 1×PBS 3 times, 5 min each time; 7) Secondary antibody incubation: Dilute the secondary antibody with horseradish peroxidase (HRP) with 1% BSA according to the instruction, add 200 μL of the secondary antibody diluent on the slide, and block at room temperature for 1 h; 8) PBS washing: Wash the slide with 1×PBS 3 times, 5 min each time; 9) Color development: Prepare the color development solution according to the instruction manual of diaminobenzidine (DAB) color development solution. Drop the DAB color development solution on the glass slide and observe the color development under the microscope. After the color development is completed, immediately place the glass slide in tap water to terminate the reaction; 10) Nuclear staining: Drop hematoxylin staining solution on the glass section and stain for 45 s, then terminate the staining with tap water.

[0068] 11) Differentiation: Place the stained section in 0.1% hydrochloric acid alcohol for 3 - 5 s for differentiation; 12) Blue return: Place the differentiated section in tap water and rinse the section gently with running water for 10 - 30 min to make it return to blue; 13) Dehydration and clearing: Immerse the stained section in 95%, 100% and 100% alcohol for 30 s respectively in the fume hood, and then immerse the section in xylene 1, xylene 2 and xylene 3 for 3 min in sequence; 14) Sealing: Take out the section from xylene 3, quickly drop 1 - 2 drops of neutral gum, cover the cover glass, and avoid forming air bubbles; dry it in the fume hood and take a photo after the section is dry.

[0069] 6. Experimental results 1) Abnormal development of the reproductive tract in adult knockout mice Compared with the control mice ( Wt1 + / flox ; Rosa + / flox ; Amhr2-Cre ), the body size of adult knockout mice ( Wt1 - / flox ; Rosa + / flox ; Amhr2-Cre ) showed no obvious abnormality ( Figure 1 in A). The knockout mice had no vaginal opening ( Figure 1 in B), no obvious fallopian tube structure was seen, the uterus did not develop, and only a thin strip - like structure remained ( Figure 1 in D and C are the uteri of control mice). H&E staining showed that this strip had no uterine lumen, uterine epithelium, endometrium and myometrium ( Figure 1 in F). The cross - section of the uterus of control mice was stained with H&E. From the inside to the outside, it was endometrial epithelium, endometrial stroma, uterine circular smooth muscle and uterine longitudinal smooth muscle in sequence ( Figure 1 in E). Primordial follicles, primary follicles, secondary follicles and corpora lutea all existed in the ovaries of knockout mice, and there was no obvious difference compared with control mice ( Figure 2 ). The uteri of mice at 5 w, 3 w and 1 d after birth had similar phenotypes to those of 8 - week - old knockout mice. Since birth, the uteri of knockout mice did not develop and presented as a thin strip - like structure, and the development of ovaries and follicles at all levels in knockout mice was not affected.

[0070] The markers FOXA1 of endometrial glands and ACTA of myometrium in the "fibrous cord tissue" of knockout mice were detected by immunohistochemical staining. The results showed that there was no expression of FOXA1 (FOXA1 antibody was purchased from abcam, ab55178) in the "uterus" of knockout mice, and only ACTA was expressed (ACTA antibody was purchased from abcam, ab11003). Figure 3 ), indicating that the "fibrous cord tissue" is a thin layer of myometrium.

[0071] 2) Mullerian duct dysplasia in knockout mice during embryogenesis Female mouse embryos at E19.5, E17.5, E15.5, E14.5, and E12.5 days were sampled, and the observation results showed Mullerian duct dysplasia in knockout mice during embryogenesis. No obvious green fluorescence was seen in the mesonephros of knockout mice at E12.5 days under the microscope. Figure 4 )

[0072] 3) The Mullerian duct of knockout mice cannot extend Wholemount staining was performed on mouse embryos throughout their lifetime. The results showed that the Mullerian duct of control mice was completely formed at E13.5 days, the marker PAX8 of Mullerian duct epithelium penetrated the entire Mullerian duct epithelium, and the expression of GFP was visible in the stromal cells of the Mullerian duct ( Figure 5 in A). However, the Mullerian duct of knockout mice could not extend, and no obvious expression of GFP was seen in the stromal cells of the Mullerian duct ( Figure 5 in B and C). This indicates that the Mullerian duct of knockout mice cannot extend.

[0073] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. Other embodiments can be obtained based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing a vagina-free uterine fallopian tube mouse model, comprising the following steps: The male Wt1 + / - Mice and females Amhr2-Cre Tool mice mate and get Wt1 + / - ;Amhr2-Cre Mice; The female Wt1 + / - ;Amhr2-Cre Mice with male conditional knockout Wt1 Genetic flox The mice were mated to obtain a vagina-free uterus and fallopian tube mouse model.

2. The method according to claim 1, characterized in that: Detection Wt1 + / - Reagents for mice include Wt1 + / - Primer set; Said Wt1 + / - The primer set includes a forward primer having a nucleotide sequence as shown in SEQ ID NO:1 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO:

2.

3. The method according to claim 2, characterized in that: Detection Wt1 + / - ;Amhr2-Cre The mouse reagents include the Wt1 + / - Primer set and Amhr2-Cre Primer set; Said Amhr2-Cre The primer set includes a forward primer having a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO:

4.

4. The method according to claim 1, characterized in that: The conditional knockout Wt1 Genetic flox Mice Rosa flox / flox Mice and Wt1 flox / flox Mice are mated.

5. The method according to claim 1, characterized in that: Detection of the conditional knockout Wt1 Genetic flox Reagents for mice include Wt1 flox / flox Primer set and Rosa flox / flox Primer set; Said Wt1 flox / flox The primer set includes a forward primer having a nucleotide sequence as shown in SEQ ID NO:5 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO:6; Said Rosa flox / flox The primer set includes a forward primer having a nucleotide sequence as shown in SEQ ID NO:7 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO:

8.

6. The method according to claim 5, characterized in that: The reagent for detecting the vagina-free uterine fallopian tube mouse model includes the Wt1 flox / flox Primer set, Rosa flox / flox Primer set, Wt1 + / - Primer set and Amhr2-Cre Primer set; Said Wt1 + / - The primer set includes a forward primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 2; Said Amhr2-Cre The primer set includes a forward primer having a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO:

4.

7. The method according to claim 1, characterized in that: The absence of uterus in the vagina-free uterine fallopian tube mouse model is manifested as at least one of the following: absence of uterine lumen, absence of uterine myometrium, and absence of endometrium.

8. The method according to claim 7, characterized in that: The absence of endometrium includes not expressing FOXA1, a marker of endometrial glands.

9. The method according to any one of claims 1 to 8, characterized in that: The ovaries of the vagina-free uterus and fallopian tube mouse model developed normally.

10. The method according to claim 9, characterized in that: The normal ovarian development is manifested as at least one of the following: normal primordial follicles, normal primary follicles, normal secondary follicles and normal corpus luteum.

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