A method for constructing a spontaneous breast cancer mouse model

Abstract

The present invention provides a method for constructing a spontaneous breast cancer mouse model, belonging to the technical field of biomedicine. The construction method of the present invention includes: hybridizing MMTV-PyMT male mice with Dnase1l3<supgt;- / -< / supgt; female mice to obtain the F1 generation; selecting F1 generation PyMT<supgt>+< / supgt>Dnase1l3<supgt>+ / −< / supgt> male mice and PyMT<supgt>−< / supgt>Dnase1l3<supgt>+ / −< / supgt> female mice for hybridization to obtain the F2 generation; selecting F2 generation PyMT<supgt>+< / supgt>Dnase1l3<supgt>- / -< / supgt> male mice and PyMT<supgt>−< / supgt>Dnase1l3<supgt>- / -< / supgt> female mice for hybridization to obtain the F3 generation female PyMT<supgt>+< / supgt>Dnase1l3<supgt>- / -< / supgt> spontaneous breast cancer mouse model. The median time for tumor formation in the spontaneous breast cancer mice obtained by the present invention is only 7-8 weeks, and the shortest time for forming breast cancer is 39 days, greatly shortening the experimental period and reducing the time and economic costs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a method for constructing a spontaneous breast cancer mouse model. Background Art

[0002] Breast cancer is a phenomenon in which breast epithelial cells undergo uncontrolled proliferation under the action of various carcinogenic factors. In the early stage of the disease, it often manifests as symptoms such as breast masses, nipple discharge, and axillary lymph node enlargement. In the late stage, distant metastasis of cancer cells can occur, leading to multi-organ lesions, directly threatening the life of the patient. Breast cancer has become the most common type of malignant tumor in women, and there is an urgent need to deeply study the occurrence and development mechanisms of breast cancer as well as treatment strategies.

[0003] Using mouse models to study the mechanisms of cancer progression and treatment regimens is currently the main preclinical approach. The spontaneous breast cancer mouse model can better simulate the laws of the occurrence and development of breast cancer in clinical patients and is a commonly used model for studying the pathogenesis and treatment methods of breast cancer. However, the median time for tumor formation in existing spontaneous breast cancer models is approximately 9 - 10 weeks, which to a certain extent hinders researchers from conducting research efficiently in a short time. Therefore, inducing the formation of breast cancer in mice in a shorter time can save the time cost of researchers and shorten the breeding time of mice, reducing the economic cost to a certain extent. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for constructing a spontaneous breast cancer mouse model, which can significantly shorten the breeding time of spontaneous breast cancer mice and reduce time and economic costs.

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

[0006] A method for constructing a spontaneous breast cancer mouse model, comprising the following steps: 1) Hybridize male MMTV-PyMT mice with Dnase1l3 - / - female mice to obtain the F1 generation; 2) Select male F1 generation PyMT + Dnase1l3 + / - mice and PyMT - Dnase1l3 + / - female mice to obtain the F2 generation; 3) Select male F2 generation PyMT + Dnase1l3 - / - mice and PyMT - Dnase1l3 - / - female mice to obtain female F3 generation PyMT + Dnase1l3 - / - spontaneous breast cancer mouse model.

[0007] Preferably, the mouse age of the hybrid mice is 10 weeks old.

[0008] Preferably, the hybridization is co-housing hybridization.

[0009] More preferably, in the co-housing hybridization, the ratio of male mice to female mice is (2-3):1.

[0010] Preferably, the spontaneous breast cancer mouse model in step 3) is female.

[0011] Preferably, the median time for tumor formation in the spontaneous breast cancer mice is 7-8 weeks.

[0012] Preferably, the time for tumor formation in the spontaneous breast cancer mice is 39-56 days.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention uses MMTV-PyMT mice to hybridize with Dnase1l3 - / - mice to obtain offspring MMTV-PyMT + Dnase1l3 - / - spontaneous breast cancer mice. The median time for tumor formation in the spontaneous breast cancer mice obtained by the present invention is only 7-8 weeks, and the shortest time for forming breast cancer is 39 days, which greatly shortens the experimental cycle and reduces the economic cost. Description of the Drawings

[0015] Figure 1 For the tumor formation time of the mice in Example 1 and Comparative Example 1;

[0016] Figure 2 For the survival curves of the mice in Example 1 and Comparative Example 1; among them, the median time for breast cancer tumor formation of 7.3 weeks is the survival curve of the PyMT + Dnase1l3 - / - female mice, and the median time for breast cancer tumor formation of 9.3 weeks is the survival curve of the PyMT + Dnase1l3 + / + female mice. Detailed Embodiments

[0017] The present invention provides a method for constructing a spontaneous breast cancer mouse model, comprising the following steps: 1) mating male MMTV-PyMT mice with Dnase1l3 - / - female mice to obtain the F1 generation; 2) selecting F1 generation PyMT + Dnase1l3 + / - male mice and PyMT - Dnase1l3 + / - female mice to obtain the F2 generation; 3) selecting F2 generation PyMT+ Dnase1l3 - / - Male mice and PyMT - Dnase1l3 - / - were mated with female mice to obtain F3 female PyMT + Dnase1l3 - / - spontaneous breast cancer mouse model.

[0018] The MMTV-PyMT mice described in the present invention are BALB / c mice, and the Dnase1l3 - / - mice are C57BL / 6 mice. The present invention does not particularly limit the sources of the MMTV-PyMT male mice and the Dnase1l3 - / - female mice, and conventional commercially available products in the art can be used. As an implementable embodiment, the MMTV-PyMT male mice and the Dnase1l3 - / - female mice described in the present invention were self-propagated by the research group of Zhu Bo in the Oncology Department of the Second Affiliated Hospital of Army Medical University.

[0019] Before the MMTV-PyMT male mice and the Dnase1l3 - / - female mice described in the present invention are mated, the MMTV-PyMT male mice need to be raised in a SPF-level animal house until 10 weeks old, and the healthy male mice with bright hair color are placed in a separate cage for 3-4 days. The Dnase1l3 - / - female mice are 10 weeks old, healthy, and have bright hair color.

[0020] The mouse age of the mating mice described in the present invention is preferably 10 weeks old. The hybridization described in the present invention is preferably cage hybridization; the ratio of male and female mice in the cage hybridization is preferably (2-3):1, more preferably 3:1.

[0021] The genotype of the F1 generation of the present invention is: PyMT + Dnase1l3 + / - and PyMT - Dnase1l3 + / - (PyMT + female mice will develop breast cancer later, so they are not used for breeding offspring). The genotype of the F2 generation of the present invention is: PyMT + Dnase1l3 - / - 、PyMT + Dnase1l3 + / - 、PyMT + Dnase1l3 + / + 、PyMT - Dnase1l3 - / - 、PyMT - Dnase1l3 + / -, PyMT - Dnase1l3 + / + .

[0022] The present invention does not have special limitations on the gender of the obtained spontaneous breast cancer mouse model. However, due to the inherent relationship between the physiological characteristics of females and breast cancer, which is worthy of further research and discussion by scientists, the spontaneous breast cancer mouse model obtained in the present invention is preferably female.

[0023] The present invention also provides a spontaneous breast cancer mouse model obtained by the above construction method. The median time for tumor formation in the spontaneous breast cancer mice of the present invention is 7 - 8 weeks; the time for tumor formation in the spontaneous breast cancer mice of the present invention is 39 - 56 days.

[0024] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0025] Example 1

[0026] Construction of a spontaneous breast cancer mouse model

[0027] MMTV-PyMT mice (male) and Dnase1l3 - / - mice (female) were independently reproduced by the research group of Zhu Bo in the Department of Oncology, the Second Affiliated Hospital of Army Medical University. Among them, the original source of MMTV-PyMT mice was transferred from the research group of Liu Xiaolong in the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences (Institute of Biochemistry and Cell Biology) to the research group of Zhu Bo. Thereafter, the scientific research achievements such as papers and patents produced by using these mice belong to the research group of Zhu Bo. In addition, MMTV-PyMT mice have been commercialized and can be purchased from the selling company. These mice were obtained by crossing MMTV-PyMT mice (male) with wild-type mice (female), and the offspring were genotyped to determine PyMT + or PyMT - , where PyMT + male mice can be used for continuous reproduction, and PyMT + female mice can develop spontaneous breast cancer. Dnase1l3 - / - mice (female) were originally transferred from the research group of Liu Xindong in the Department of Pathology, the First Affiliated Hospital of Army Medical University to the research group of Zhu Bo. The two parties agreed that any economic benefits or patent ownership brought by the research on tumors using these mice belong to the research group of Zhu Bo. These mice can be obtained by crossing male and female Dnase1l3 + / - mice, and the offspring were genotyped to obtain Dnase1l3 - / - mice (female). The feeding, reproduction, and experimental operations of the above mice have passed the review of the Experimental Animal Welfare and Ethics Review Committee of the Third Military Medical University (Army Medical University) (AMUWEC2020376).

[0028] (1) Obtaining F1 generation: MMTV-PyMT (male) mice were raised in a specific pathogen free (SPF) animal house until 10 weeks of age. First, healthy male mice with shiny hair were placed in a separate cage for 3 - 4 days, and then mated with 10-week-old, healthy, shiny-haired Dnase1l3 - / - female mice. The mating was carried out in the evening, with a male-to-female ratio of 3:1. The next morning (about 12 hours after mating), the formation of vaginal plugs in female mice was checked. The mice with vaginal plugs were placed in separate cages and waited for delivery.

[0029] When the F1 generation grew to 6 - 8 weeks old, their toe tips were taken for genomic DNA extraction. PCR reactions were performed according to the identification primers of the two parental mice to obtain the genotypes of the F1 generation: PyMT + Dnase1l3 + / - and PyMT - Dnase1l3 + / - (Note: Female PyMT + mice will develop breast cancer later, so they are not used for breeding offspring).

[0030] (2) Obtaining F2 generation: Select 10-week-old PyMT + Dnase1l3 + / - male mice of the F1 generation and 10-week-old PyMT - Dnase1l3 + / - female mice of the F1 generation, and carry out cage mating and hybridization in the aforementioned manner to obtain the F2 generation, with genotypes: PyMT + Dnase1l3 - / - 、PyMT + Dnase1l3 + / - 、PyMT + Dnase1l3 + / + 、PyMT - Dnase1l3 - / - 、PyMT - Dnase1l3 + / - 、PyMT - Dnase1l3 + / + 。

[0031] (3) Obtaining F3 generation: Select 10-week-old PyMT + Dnase1l3 - / - male mice and 10-week-old PyMT - Dnase1l3 - / - female mice of the F2 generation for cage mating and hybridization to obtain PyMT + Dnase1l3 - / -Spontaneous breast cancer female mice.

[0032] Comparative Example 1

[0033] The specific construction method is the same as that of Example 1, except that the F3 generation is obtained in step (3) as follows:

[0034] Select 10-week-old PyMT + Dnase1l3 + / + male mice and 10-week-old PyMT - Dnase1l3 + / + female mice were caged and hybridized to obtain PyMT + Dnase1l3 + / + female mice, that is, the commonly used spontaneous breast cancer mice.

[0035] Experimental Example 1

[0036] Statistically analyze the PyMT + Dnase1l3 - / - spontaneous breast cancer female mice and PyMT in Comparative Example 1 + Dnase1l3 + / + The median tumorigenesis time and breast cancer formation time of female mice with breast cancer. The specific results are shown in Table 1 Figure 1-2 as follows.

[0037] The median tumorigenesis time of breast cancer is the time when 50% of the individuals form tumors; the breast cancer formation time is the time when a significantly palpable subcutaneous tumor mass forms in the mammary gland of the mouse starting from the date of birth of the mouse. During the statistical process, the growth environment, feed, etc. of each group of mice (9 mice in each group) were exactly the same.

[0038] Table 1 Breast cancer formation time of mice in Example 1

[0039]

[0040]

[0041] From Table 1 and Figure 1-2 the data, it can be seen that compared with PyMT + Dnase1l3 + / + spontaneous breast cancer female mice in Comparative Example 1, the PyMT + Dnase1l3 - / - female mice in the present invention can advance the tumorigenesis time by about 18 days and the breast cancer formation time by about 2 weeks. Moreover, the shortest breast cancer tumorigenesis time of the PyMT + Dnase1l3 - / - female mice in the present invention is only 39 days, which can greatly shorten the breeding time of spontaneous breast cancer mice and reduce the economic cost.

[0042] 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 spontaneous breast cancer mouse model, characterized in that, it comprises the following steps: 1) Cross MMTV-PyMT male mice with Dnase1l3 - / - female mice to obtain the F1 generation; 2) Select F1 generation PyMT + Dnase1l3 + / - male mice and PyMT - Dnase1l3 + / - female mice were crossed to obtain the F2 generation; 3) Select F2 generation PyMT + Dnase1l3 - / - Male mice and PyMT - Dnase1l3 - / - Hybridize with female mice to obtain F3 generation female PyMT + Dnase1l3 - / - Spontaneous breast cancer mouse model.

2. According to the construction method described in claim 1, characterized in that, The MMTV-PyMT male mice, Dnase1l3 - / - female mice, F1 generation PyMT + Dnase1l3 + / - male mice and PyMT - Dnase1l3 + / - female mice, F2 generation PyMT + Dnase1l3 - / - male mice and PyMT - Dnase1l3 - / - The age of all female mice is 10 weeks old.

3. According to the construction method described in claim 1, characterized in that, the hybridization in steps 1), 2), and 3) is co-housing hybridization.

4. According to the construction method described in claim 3, characterized in that, in the co-housing hybridization, the ratio of male to female mice is (2-3):

1.

5. According to the construction method described in any one of claims 1-4, characterized in that, the median time for tumor formation in the spontaneous breast cancer mice is 7-8 weeks.

6. According to the construction method described in any one of claims 1-4, characterized in that, the time for tumor formation in the spontaneous breast cancer mice is 39-56 days.

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