A method for constructing a mouse model of vagina-free uterus and fallopian tubes

Abstract

The present invention provides a method for constructing a mouse model without vagina, uterus and oviduct, belonging to the field of biotechnology. The present invention provides a method for constructing a mouse model without vagina, uterus and oviduct, which comprises mating Wt1 <supgt; + / ‑ < / supgt; mice with Amhr2-Cre tool mice, and mating the obtained Wt1 <supgt; + / ‑ < / supgt; ; Amhr2-Cre mice with Wt1 gene flox conditional knockout Wt1 gene in mice, so as to specifically knockout the Wt1 gene in the genome of mouse Müllerian duct interstitial cells, and obtain a mouse model without vagina, uterus and oviduct with stable phenotype and high penetrance. The method of the present invention provides an important bridge for the drug research and development for treating female genital tract malformations and the clinical disease treatment.

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 vagina-uterus-fallopian tube absence. 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 helps to better study the pathogenesis, mechanism of MRKH syndrome and the screening of related drugs. The unique comprehensive advantages of gene-edited mouse models in model organisms are not only manifested in the genetic and physiological similarities with 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 helps to deeply reveal the action mechanism of potential pathogenic genes, provides a tool for preclinical evaluation of drugs, and becomes an important bridge for the effective transformation from basic research on drug development to clinical disease treatment applications. 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 vagina-uterus-fallopian tube absence, 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:

[0006] The present invention provides a method for constructing a mouse model of vagina-uterus-fallopian tube absence, comprising the following steps:

[0007] Mating male Wt1 + / - mice with female Amhr2-Cre tool mice to obtain Wt1 + / - ; Amhr2-Cre mice;

[0008] Mating female Wt1 + / - ; Amhr2-Cre mice with male conditional knockout Wt1 gene flox mice to obtain a mouse model of vagina-uterus-fallopian tube absence.

[0009] Preferably, detecting theWt1 + / - Reagents for mice include Wt1 + / - A primer set;

[0010] 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.

[0011] Preferably, for detecting the Wt1 + / - ; Amhr2-Cre Reagents for mice include the Wt1 + / - primer set and Amhr2- Cre a primer set;

[0012] The Amhr2-Cre The 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.

[0013] Preferably, the Wt1 gene flox of the Rosa flox / flox mice is obtained by mating Wt1 flox / flox mice with

[0014] Preferably, for detecting the Wt1 gene flox of the Wt1 flox / flox reagents for mice include Rosa flox / flox a primer set and

[0015] 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;

[0016] 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.

[0017] Preferably, for detecting the reagent for the mouse model without vagina, uterus and oviduct includes the Wt1 flox / flox primer set, the Rosa flox / floxPrimer set, Wt1 + / - primer set and Amhr2-Cre primer set;

[0018] 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;

[0019] 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.

[0020] Preferably, in the mouse model without vagina, uterus and oviduct, the absence of the uterus is manifested as at least one of the following: no uterine lumen, no myometrium and no endometrium.

[0021] Preferably, the absence of the endometrium includes the non-expression of the endometrial gland marker FOXA1.

[0022] Preferably, the ovaries of the mouse model without vagina, uterus and oviduct develop normally.

[0023] 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 corpora lutea.

[0024] The present invention has the following advantages compared with the prior art:

[0025] The present invention provides a method for constructing a mouse model without vagina, uterus and oviduct. A male Wt1 + / - mouse is mated with a female Amhr2-Cre tool mouse to obtain Wt1 + / - ; Amhr2-Cre a mouse; the female Wt1 + / - ; Amhr2-Cre mouse is mated with a male mouse with conditional knockout of Wt1 a gene to obtain a mouse model without vagina, uterus and oviduct. The method of the present invention knocks out flox a gene in the genomic DNA of the Müllerian duct stromal cells of the mouse, Wt1Gene, a phenotypically stable vagina-free uterine and fallopian tube mouse model can be obtained. The embodiment of the present invention compares the reproductive tract development of knockout mice and control mice. The results show that compared with the control mice, the knockout mice have no vaginal opening, the uterus is not developed, and there is a thin layer of uterine muscle layer, and no obvious fallopian tube structure. In addition, the method provided by the present invention does not affect the size of the knockout mice, nor does it affect the development of their ovaries and follicles at all levels, providing an important bridge for the development of drugs for the treatment of female reproductive tract malformations and clinical disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The morphological comparison of the external genitalia and uterus of adult (8 weeks) knockout mice and control mice; A: comparison of mouse body size; B: comparison of mice without vaginal opening; C: morphology of reproductive organs of control mice; D: morphology of reproductive organs of knockout mice; E: H&E staining results of cross-section of uterus of control mice; F: H&E staining results of cord-like structures of knockout mice;

[0027] Figure 2 The morphological diagrams of the ovaries (3 weeks) of knockout mice and control mice; A and C are the ovaries of control mice; B and D are the ovaries of knockout mice;

[0028] Figure 3 The diagrams show the expression results of endometrial gland and myometrial markers in knockout mice and control mice; A: expression results of ACTA in the uterus of control mice; B: expression results of ACTA in the uterus of knockout mice; C: expression results of FOXA1 in the uterus of control mice; D: expression results of FOXA1 in the uterus of knockout mice;

[0029] Figure 4 The color images of the urogenital system of E12.5-day mouse embryos under a fluorescence microscope; A and B are images of control mice, C and D are images of knockout mice, and O in A to D represents ovary, and M represents mesonephros;

[0030] Figure 5 Wholemount staining of the Mullerian duct of E13.5 day embryonic mice; A: Wholemount staining of the mesonephros of control mice, B and C: Wt1 Wholemount staining results of the mesonephros of knockout mice, PAX8 marks the Mullerian duct epithelium, and GFP marks the Mullerian duct interstitial cells. DETAILED DESCRIPTION

[0031] The present invention provides a method for constructing a vagina-free uterine fallopian tube mouse model, comprising the following steps:

[0032] The male Wt1 + / -The mouse is mated with a female Amhr2-Cre tool mouse to obtain Wt1 + / - ; Amhr2-Cre a mouse;

[0033] The female Wt1 + / - ; Amhr2-Cre mouse is mated with a male Wt1 mouse with conditional knockout of the flox gene to obtain a mouse model without vagina, uterus and fallopian tubes.

[0034] In the present invention, the preparation method of the Wt1 + / - mouse preferably utilizes the principle of gene recombination. The replacement vector pWT has a 10 kb homologous domain with the Wt1 gene. The first exon of the Wt1 gene and the 0.5 kb sequence upstream thereof are replaced through the homologous domain in the replacement vector pWT; PGK-neo-poly(A) The thymidine kinase, TK gene is used for positive selection, and the thymidine kinase ( Wt1 + / - The specific preparation method of the mouse refers to the prior art (Kreidberg JA, Sariola H, Loring JM, et al. WT-1 is required forearly kidney development. Cell.1993;74:679-91.). In the method of the present invention, the introduction of the Wt1 + / - mouse 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 mouse, thereby obtaining a mouse model without vagina, uterus and fallopian tubes.

[0035] In the present invention, the reagent for detecting the Wt1 + / - mouse 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 using theWt1 + / - When the primer set is amplified to obtain an amplification product of 250 bp, the mouse to be tested is Wt1 + / - a heterozygote. When no amplification product of 250 bp is obtained, the mouse to be tested is Wt1 + / + a homozygote.

[0036] After obtaining Wt1 + / - the mouse, the present invention mates the male Wt1 + / - mouse with a female Amhr2-Cre tool mouse to obtain Wt1 + / - ; Amhr2-Cre a mouse.

[0037] In the present invention, Amhr2-Cre the preparation method of the tool mouse is preferably to use the gene targeting method to introduce a DNA fragment of a Cre -neomycin (neo) sequence into mouse embryonic stem cells, and this fragment is integrated with the mouse chromosome through the principle of homologous recombination. Cre-neo The sequence is inserted into the 5th exon of the Amhr2 gene of the mouse. Amhr2-Cre The specific preparation method of the tool mouse is preferably referred 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.).

[0038] In the present invention, the male Wt1 + / - mouse is mated with a female Amhr2-Cre tool mouse, and after genotype identification and screening, Wt1 + / - ; Amhr2-Cre a mouse is obtained. The reagents for detecting the Wt1 + / - ; Amhr2-Cre mouse 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, with the Wt1+ / - The primer set was amplified to obtain an amplification product of 250 bp, and using the Amhr2-Cre primer set for amplification, an amplification product of 550 bp was obtained. The mouse to be tested is Wt1 + / - ; Amhr2-Cre a mouse.

[0039] Obtained Wt1 + / - ; Amhr2-Cre After obtaining the mouse, the present invention preferably uses female Wt1 + / - ; Amhr2-Cre mice and male conditional knockout Wt1 gene flox mice for mating to obtain a mouse model without vagina, uterus and fallopian tubes.

[0040] 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 after genotype identification and screening, Wt1 + / flox ; Rosa + / flox mice are obtained; Wt1 + / flox ; Rosa + / flox mice are mated with Wt1 + / flox ; Rosa + / flox mice, and after genotype identification and screening, the conditional knockout Wt1 gene flox mice ( Wt1 flox / flox ; Rosa flox / flox ) are obtained. The Rosa flox / flox mice are a kind of double-fluorescent Cre reporter mice, which are mediated by Cre and LoxPBefore excision, membrane-targeted Tomato (mT) is expressed, and after excision, membrane-targeted green fluorescent protein (mG) is expressed. When there is no Cre enzyme, the pCA promoter (chicken β-actin core promoter with a CMV enhancer) drives the expression of membrane-localized Tomato, which terminates at the first pA sequence (terminator); when there is Cre enzyme, Cre after recombination is mediated, the mT + pA sequence is excised, enabling the pCA promoter to drive the expression of green fluorescent protein (GFP). Rosa flox / flox The specific method for preparing the mice is preferably referred 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 method for preparing the mice is preferably to insert a Wt1 site on both sides of exons 8 and 9 of the gene in mouse embryonic cells using the principle of gene recombination. Exons 8 and 9 encode LoxP the second and third zinc finger binding domains of the Wt1 protein, and the latter two are Wt1 key structural domains for the Wt1 flox / flox The specific method for preparing the mice is referred 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.).

[0041] In the present invention, the reagent for detecting the Wt1 gene in the flox conditionally knockout mice preferably includes Wt1 flox / flox a primer set and Rosaflox / 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. Using the genome of the mouse to be tested as a template and amplifying with the Wt1 flox / flox primer set, only an amplification product of 150 bp is obtained, indicating that the mouse to be tested is Wt1 + / + a homozygote; when only an amplification product of 250 bp is obtained, it indicates that the mouse to be tested is Wt1 flox / flox a homozygote, and when an amplification product of 150 bp and an amplification product of 250 bp are obtained simultaneously, it indicates that the mouse to be tested is Wt1 + / flox a heterozygote. Using the genome of the mouse to be tested as a template and amplifying with the Rosa flox / flox primer set, only an amplification product of 250 bp is obtained, indicating that the mouse to be tested is Rosa flox / flox a homozygote, and when only an amplification product of 330 bp is obtained, it indicates that the mouse to be tested is Rosa + / + a homozygote, and when an amplification product of 250 bp and an amplification product of 330 bp are obtained simultaneously, it indicates that the mouse to be tested is Rosa + / flox a heterozygote. Using the genome of the mouse to be tested as a template and amplifying with the Wt1 flox / flox primer set, only an amplification product of 250 bp is obtained, and using the Rosa flox / flox primer set to amplify, only an amplification product of 330 bp is obtained, and the mouse to be tested is a mouse with conditional knockout of the Wt1 gene ( flox 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, theWt1 + / - primer sets and the Amhr2-Cre primer sets. Using the genomic DNA of the mouse to be tested as a template, respectively using Wt1 flox / flox primer set, the Rosa flox / flox primer set, the Wt1 + / - primer set and the Amhr2-Cre primer set for amplification, and amplification products of 250bp, 250bp, 250bp and 550bp are obtained, indicating that the mouse to be tested is Wt1 - / flox ; Rosa + / flox ; Amhr2-Cre.

[0042] In the present invention, the Wt1 + / - primer set, Amhr2-Cre primer set, Wt1 flox / flox primer set and Rosa flox / flox The reaction system of the PCR of the primer set is preferably independently 2×Taq DNA polymerase 10 μL, upstream primer 1 μL, downstream primer 1 μL, template 1 μL, double distilled water (ddH2O) 7 μL. 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.

[0043] 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 weeks, more preferably 9 weeks. 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, and defining the sex and age of the mating mice is beneficial to obtaining a mouse model without vagina, uterus and oviduct with stable phenotype and high penetrance. The method of the present invention has been verified by repeated experiments, and the obtained mouse model without vagina, uterus and oviduct has a stable phenotype and high penetrance.

[0044] In the present invention, in the mouse model of absence of vagina, uterus and fallopian tubes constructed by the above method, the absence of uterus preferably mainly shows at least one of the following: absence of uterine lumen, absence of myometrium 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.

[0045] The method of the present invention can specifically knockout the Wt1 gene in the genome of mouse Müllerian duct interstitial cells to obtain a mouse model of absence of vagina, uterus and fallopian tubes with stable phenotype 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, showing a thin cord-like structure, without uterine lumen, uterine epithelium, endometrium and myometrium, and no obvious fallopian tube structure was seen. There was no obvious abnormality in physical size, and there was no obvious abnormality in primordial follicles, primary follicles, secondary follicles and corpus luteum. The uteri of mice at 5w, 3w and 1d after birth were similar in phenotype to those of 8w knockout mice, indicating that the uteri of knockout mice did not develop from birth, and the development of ovaries and follicles at all levels in knockout mice was not affected. Microscopic observation of knockout mouse embryos showed that the Müllerian ducts of knockout mouse embryos were underdeveloped. Whole-mount staining was performed on mouse embryos throughout their lifetime, and the results showed that the Müllerian ducts of knockout mice could not extend.

[0046] To further illustrate the present invention, the following describes in detail a method for constructing a mouse model of absence of vagina, uterus and fallopian tubes provided by the present invention with reference to the drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0047] 1. Experimental animals

[0048] All mice used in the present invention are 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 feeding of experimental animals follows the "Regulations on the Administration of Experimental Animals" in Beijing and the regulations of animal health care institutions. The feeding environment is 12h light and 12h dark alternating every day, the temperature is maintained at 24±1°C, and the humidity is kept at about 35±4%. Animal experiments are strictly carried out in accordance with the regulations of the Animal Ethics Committee.

[0049] (1) Amhr2-CreThe 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.).

[0050] (2) Rosa flox / flox The preparation method of the 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..

[0051] (3) Wt1 flox / flox Mice: Using the principle of gene recombination in mouse embryonic cells Wt1 Insert one on each side of the 8th and 9th exons of the gene LoxP The 8th and 9th exons encode Wt1 The 2nd and 3rd 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 preparation method of the 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.).

[0052] (4) Wt1 + / - Mice: By constructing a replacement vector pWT, which has a 10kb homologous domain with the Wt1 gene; Using the principle of gene recombination to replace the first exon of the Wt1 gene and the 0.5kb sequence upstream of it, PGK-neo-poly(A) The gene is used for positive selection, and thymidine kinase ( thymidine kinase, TKThe gene is used for negative selection. pWT was introduced into the Jl embryonic stem cells cell line. After 8 days of screening, clones resistant to G418 and FIAU were selected. The selected clones were injected into C57BL / 6 blastocysts, and the resulting chimeric mice were crossed with wild-type C57BL / 6 mice, and the mutation was transmitted through the germline. 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.).

[0053] 2. The experimental instruments and their corresponding manufacturers are shown in Table 1.

[0054] Table 1 Experimental Instruments

[0055]

[0056] 3. Reagent Information

[0057] (1) The experimental reagents and their corresponding reagent companies are shown in Table 2.

[0058] Table 2 Experimental Reagents

[0059]

[0060] (2) Preparation of commonly used experimental reagents

[0061] Preparation of 4% PFA-PBS buffer (1000 ml): Weigh 40 g of paraformaldehyde (PFA), 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, and after preparation, it can be aliquoted and stored at -20°C.

[0062] (3) The antibodies, their usage concentrations, and the corresponding reagent companies are shown in Table 3.

[0063] Table 3 Antibody Information

[0064]

[0065] Example 1

[0066] Preparation Method of Mouse Model without Vagina, Uterus, and Oviduct

[0067] 1. Mouse breeding strategy

[0068] (1) Mate male Wt1 + / - mice with female Amhr2-Cre tool mice, and screen to obtain Wt1 + / - ; Amhr2-Cre mice;

[0069] (2) Mate Rosa flox / flox mice with Wt1 flox / flox mice, and screen to obtain Wt1 + / flox ; Rosa + / flox mice; Mate Wt1 + / flox ; Rosa + / flox mice with Wt1 + / flox ; Rosa + / flox mice, and screen to obtain mice with conditional knockout of Wt1 gene flox ( Wt1 flox / flox ; Rosa flox / flox );

[0070] (3) Mate female Wt1 + / - ; Amhr2-Cre mice with male Wt1 flox / flox ; Rosa flox / flox to screen and obtain knockout mice with knockout of Wt1 gene in mouse Müllerian stromal cells ( Wt1 - / flox ; Rosa + / flox ; Amhr2- Cre ) as a mouse model without vagina, uterus and fallopian tubes; Obtain Wt1 + / flox ; Rosa + / flox ; Amhr2-Cre mice as control mice.

[0071] When breeding mice, introduce Wt1 + / - to avoid the problem of incomplete knockout caused by low efficiency of Cre mice, CreThe enzyme only needs to knock out two Flox sites on one sister chromatid. Rosa flox / flox Mouse can be marked Cre Positive cells.

[0072] 2. Mating mice and picking up bolts

[0073] Between 16:00 and 18:00 the previous afternoon, female and male mice of reproductive age (9 weeks) were caged together, with the ratio of female to male mice being 2:1. Sufficient mouse food and water were prepared, and the mouse number, genotype, sex, date of birth and cage-mixing date were marked in detail on the breeding card.

[0074] The plug was removed between 8 and 10 a.m. on the second day. The vaginal opening of the female mouse was gently opened with tweezers to observe whether there was a milky white solid substance. 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 into cages, and mating continued in the afternoon of the same day, and the plug was removed the next day.

[0075] 3. Mouse Labeling

[0076] Mice can be numbered and marked with ear tags 2 weeks after birth. Gently grasp the skin of the mouse's back and neck with your left hand and fix it in the palm of your hand. Clamp the mouse's tail with the little finger and ring finger of your left hand; arrange the ear tags in order, hold the ear tag forceps in your right hand and gently clamp the ear tag, quickly clamp the ear tag to the mouse's right ear, and check whether the ear tag is clamped tightly. Use ophthalmic scissors to gently cut off the mouse's tail about 5mm at the end, place it in a 1.5ml EP tube, and mark the tube with the number corresponding to the ear number. For mice within 2 weeks after birth, if genotype identification is urgently needed, use the method of clipping the toes to mark them.

[0077] 4. Mouse genotype identification

[0078] Take the tail or toe of the mouse to be identified and put it into a 1.5mL EP tube, add 99μL lysis solution and 1μL 20mg / mL proteinase K, place it on a shaker, and lyse at 56℃ 270rpm for at least 4h. Then use a 95℃ metal bath to inactivate proteinase K for later use, or temporarily store it in a 4℃ refrigerator.

[0079] The above cleavage products were used as templates to perform polymerase chain reaction (PCR) and then perform genotype identification.

[0080] The primer sequences, annealing temperatures and corresponding band sizes for mouse genotype identification are shown in Table 4 .

[0081] Table 4 Primer sequences, annealing temperatures and band sizes for mouse genotype identification

[0082]

[0083] Note: F: upstream primer; R: downstream primer; R1: downstream primer of wild-type strand; R2: downstream primer of strand containing LoxP site.

[0084] When performing PCR reaction for mouse genotype identification, the reaction system is as follows: 2×Taq DNA polymerase: 10 μL, upstream primer: 1 μL, downstream primer: 1 μL, template: 1 μL, double-distilled water (ddH2O): 7 μL.

[0085] The PCR reaction procedure is as follows:

[0086] 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.

[0087] Gel preparation: Prepare a 2% agarose gel with 1×TAE solution.

[0088] Electrophoresis imaging: Use 1×TAE solution as the electrophoresis buffer; load samples in order, add 10 μL of PCR product to each well, add at least 6 μL of Marker to 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. Voltage: 100 v, electrophoresis time: 30 min, image under a gel imager.

[0089] Example 2

[0090] Mouse genital tract detection

[0091] 1. Mouse genital tract sampling

[0092] Euthanize the mouse by cervical dislocation. Place the knockout mouse and its littermate control mouse side by side and take a photo (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, and 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.

[0093] Place the isolated female mouse reproductive system 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 mouse and its littermate control mouse side by side and take a photo for record.

[0094] 2. Tissue fixation

[0095] Fix in 4% PFA phosphate buffer solution overnight. Wash 3 times with 1×PBS, then place in 70% alcohol. If not embedded immediately, it can be temporarily stored in a 4°C refrigerator after being placed in 70% alcohol.

[0096] 3. Paraffin embedding

[0097] 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.

[0098] 2) Gradient alcohol dehydration: Immerse the embedding cassette in 70%, 80%, and 90% alcohol at room temperature for 1 h respectively. During the immersion process, gently shake on a shaker to fully dehydrate the tissue. Then place it in 100% alcohol at room temperature for 40 min × 2 times.

[0099] 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 put it into xylene at room temperature for 30 min.

[0100] 4) Wax infiltration: Place the paraffin in the oven the day before the experiment to melt the wax at 60°C to ensure sufficient wax liquid on the day of the experiment. Place the embedding cassette successively in paraffin I, paraffin II, and paraffin III at 70°C for 1 h each.

[0101] 5) Embedding: Turn on the embedding box to melt the wax 3 h before embedding. 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 to solidify. The solidified tissue is stored in a 4°C refrigerator and waiting for sectioning.

[0102] 4. Hematoxylin-eosin staining (HE staining)

[0103] 1) Deparaffinization: Immerse the paraffin sections successively in xylene 1, xylene 2, and xylene 3 in a fume hood for 10 min each;

[0104] 2) Gradient alcohol rehydration: Immerse successively in 100%, 100%, 100%, 95%, 80%, and 70% alcohol for 5 min each;

[0105] 3) Washing: Wash 3 times with PBS, 5 min each time;

[0106] 4) Hematoxylin staining: Drop hematoxylin staining solution on the glass section and stain for 45 s, and terminate the staining with tap water;

[0107] 5) Differentiation: Place the stained section in 0.1% hydrochloric acid alcohol for differentiation for 3 - 5 s;

[0108] 6) Blueing: Place the differentiated sections in tap water and rinse the sections gently under running water for 10 - 30 min to blue them.

[0109] 7) Eosin staining: Prepare a working solution of eosin stain by mixing eosin stain with 95% alcohol at a ratio of 1:1, and stain for 30 s (the time can be adjusted according to the staining situation). After staining, wash with tap water for 5 min, observe at any time, and then place in 95% alcohol.

[0110] 8) Dehydration and clearing: Immerse the stained sections in 95%, 100%, and 100% alcohol for 30 s in sequence in a fume hood, and then immerse the sections in xylene 1, xylene 2, and xylene 3 for 3 min in sequence.

[0111] 9) Mounting: Take out the sections from xylene 3, quickly drop 1 - 2 drops of neutral balsam, cover with a cover slip, and avoid forming air bubbles. Air dry in a fume hood and take pictures after the sections are dry.

[0112] 5. Immunofluorescence staining of paraffin sections

[0113] 1) Steps 1 - 3 refer to the HE staining of paraffin sections.

[0114] 2) Antigen retrieval: Place the sections in a citrate antigen retrieval solution and boil in a microwave oven at high power (about 5 min); immediately switch to low power and continue boiling for 15 min after boiling; cool naturally to room temperature.

[0115] 3) PBS washing: Wash the sections with 1×PBS 3 times, 5 min each time.

[0116] 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.

[0117] 5) Primary antibody incubation: Dilute the blocking solution with 0.3% TritonX - 100 to 1% BSA, which is used as the primary antibody diluent. Dilute the antibody according to the antibody usage concentration in the instruction manual. Pour out the blocking solution on the sections, shake off as much as possible, and add 200 μL of the primary antibody diluent, and block at room temperature for 1 h.

[0118] 6) PBS washing: Wash the sections with 1×PBS 3 times, 5 min each time.

[0119] 7) Secondary antibody incubation: Dilute the fluorescent secondary antibody with 1×PBS according to the instruction manual, add DAPI, and incubate at room temperature for 1 h in the dark.

[0120] 8) PBS washing: Wash the sections with 1×PBS 3 times, 5 min each time, in the dark.

[0121] 9) Mounting: Take out the sections from PBS, quickly add 1 - 2 drops of anti - fluorescence quenching agent, cover with a coverslip, and avoid forming air bubbles. Place the slides in a dark box and store temporarily at 4°C, and observe under a fluorescence microscope as soon as possible.

[0122] 5. Immunohistochemical staining of paraffin sections

[0123] 1) Steps 1 - 4 refer to the immunofluorescence staining of paraffin sections;

[0124] 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;

[0125] 3) Primary antibody incubation: Dilute the blocking solution to 1% BSA with 1×PBS, and the latter is used as the primary antibody diluent. Dilute the antibody according to the antibody usage concentration in the instruction manual. Pour out the blocking solution on the section, shake off as much as possible, and add 200 μL of the primary antibody diluent, then block at room temperature for 1 h;

[0126] 4) PBS washing: Wash the sections 3 times with 1×PBS, 5 min each time;

[0127] 5) Remove peroxidase in the tissue: Immerse the sections in 3% H2O2 in the dark for 10 min;

[0128] 6) PBS washing: Wash the sections 3 times with 1×PBS, 5 min each time;

[0129] 7) Secondary antibody incubation: Dilute the secondary antibody with horseradish peroxidase (HRP) according to the instruction using 1% BSA, add 200 μL of the secondary antibody diluent on the section, and block at room temperature for 1 h;

[0130] 8) PBS washing: Wash the sections 3 times with 1×PBS, 5 min each time;

[0131] 9) Chromogenic reaction: Prepare the chromogenic solution according to the instruction manual of diaminobenzidine (DAB) chromogenic solution. Drop the DAB chromogenic solution on the slide, observe the chromogenic situation under the microscope, and immediately put the slide into tap water to terminate the reaction after the chromogenic reaction is completed;

[0132] 10) Nuclear staining: Drop hematoxylin staining solution on the glass slide and stain for 45 s, then terminate the staining with tap water.

[0133] 11) Differentiation: Place the stained sections in 0.1% hydrochloric acid alcohol for 3 - 5 s for differentiation;

[0134] 12) Blueing: Place the differentiated sections in tap water and rinse the sections gently with running water for 10 - 30 min to make them blue;

[0135] 13) Dehydration and clearing: In the fume hood, sequentially immerse the stained sections in 95%, 100%, and 100% alcohol for 30 s each, and then immerse the sections in xylene 1, xylene 2, and xylene 3 for 3 min each.

[0136] 14) Mounting: Take out the sections from xylene 3, quickly drop 1 - 2 drops of neutral balsam, cover with a coverslip, and avoid forming air bubbles; dry in the fume hood, and take pictures after the sections are dry.

[0137] 6. Experimental results

[0138] 1) Abnormal development of the reproductive tract in adult knockout mice

[0139] 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). For the cross - section of the uterus of control mice stained with H&E, from the inside to the outside, there were 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 a phenotype similar to that of 8 - w knockout mice. Since birth, the uterus of knockout mice did not develop and was a thin strip - like structure, and the development of the ovaries and follicles at all levels in knockout mice was not affected.

[0140] Detect the markers FOXA1 of endometrial glands and ACTA of myometrium in the "fibrous strip tissue" of knockout mice 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 (ACTA antibody was purchased from abcam, ab11003) was expressed ( Figure 3 ), indicating that this "fibrous strip tissue" was a thin layer of myometrium.

[0141] 2) Mullerian duct dysplasia in knockout mice during embryogenesis

[0142] Female mouse embryos at E19.5, E17.5, E15.5, E14.5, and E12.5 were collected, and the observation results showed Mullerian duct dysplasia in knockout mice during embryogenesis. No obvious green fluorescence was observed in the mesonephros of knockout mice at E12.5 under the microscope ( Figure 4 ).

[0143] 3) The Mullerian duct of knockout mice cannot extend

[0144] Wholemount staining was performed on mouse embryos throughout embryogenesis. The results showed that the Mullerian duct of control mice was completely formed at E13.5. The marker PAX8 of the Mullerian duct epithelium ran through the entire Mullerian duct epithelium, and GFP expression was visible in the Mullerian duct stromal cells ( Figure 5 in A). However, the Mullerian duct of knockout mice could not extend, and no obvious GFP expression was observed in the Mullerian duct stromal cells ( Figure 5 in B and C). This indicates that the Mullerian duct of knockout mice cannot extend.

[0145] 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 embodiments. 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 mouse model of vagina, uterus and fallopian tube deficiency, comprising the following steps: Mate male Wt1 + / - mice with female Amhr2-Cre tool mice to obtain Wt1 + / - ; Amhr2-Cre mice; Female Wt1 + / - ; Amhr2-Cre mice were mated with male mice with conditional knockout of Wt1 gene to obtain a mouse model without vagina, uterus and oviduct. flox ​ 2. The method according to claim 1, characterized in that The reagent for detecting the Wt1 + / - mouse includes Wt1 + / - a 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.

3. The method according to claim 2, wherein Detect the Wt1 + / - ; Amhr2-Cre reagents for mice include the Wt1 + / - primer sets and Amhr2-Cre primer sets; 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.

4. The method according to claim 1, wherein The conditional knockout Wt1 of the flox gene was obtained by mating Rosa flox / flox mice with Wt1 flox / flox mice.

5. The method according to claim 1, wherein Detecting the conditional knockout Wt1 of the flox gene in mice includes Wt1 flox / flox a primer set and Rosa flox / flox a primer set; The Wt1 flox / flox The 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 The 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.

6. The method according to claim 5, characterized in that, The reagents for detecting the vagina-free uterus and fallopian tube mouse model include the Wt1 flox / flox primer set, the Rosa flox / flox primer set, Wt1 + / - primer set and Amhr2-Cre 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; 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.

7. The method according to claim 1, wherein In the mouse model of vagina, uterus and fallopian tube deficiency, the absence of uterus is manifested as at least one of the following: no uterine lumen, no myometrium, and no endometrium.

8. The method according to claim 7, wherein The absence of endometrium includes the non-expression of 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 mouse model of vagina, uterus and fallopian tube deficiency develop normally.

10. The method according to claim 9, wherein 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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