Method for constructing primary cardiac tumor mouse model

Abstract

The invention belongs to the technical field of biology, and particularly relates to a construction method of a mouse model for cardiac primary tumor. Comprising the following steps: (1) breeding Scgb1a1flox / + mice with Scgb1a1 conditionally knocked out to obtain Scgb1a1flox / flox homozygous mice, wherein the Scgb1a1flox / flox homozygous mice are regarded as F0.5 generation; (2) breeding the F0.5 generation and a Myh6Cre + tool mouse, and screening out a progeny of Scgb1a1flox / +-Myh6Cre + as an F1 generation; (3) screening out offspring of Scgb1a1flox / flox-Myh6Cre + after breeding of the F1 generation, wherein the offspring is regarded as an F2 generation; (4) after the F2 generation continues to breed, screening out offspring of Scgb1a1flox / flox-Myh6Cre +, wherein the offspring is regarded as an F2 + 1 generation; and obtaining the heart primary tumor mouse model from the F2 + 1 generation. The construction method does not need any physical and chemical induction to mice.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for constructing a primary cardiac tumor mouse model. Background Art

[0002] Mouse tumor models play an important role in oncology research. Their easy availability, short growth cycle, and ease of operation make mice a commonly used tumor model animal. Mouse tumor models can simulate the growth process of human tumors and provide an important experimental platform for studying the occurrence, development, and treatment of tumors.

[0003] At present, the methods for constructing tumor models mainly include spontaneous or induced tumor models, which mainly rely on the tumor incidence of the experimental animals themselves or the administration of carcinogens to induce tumors. The spontaneous rate of primary cardiac tumors is extremely low, and there are no reports of spontaneous primary cardiac tumors in adult mice at home and abroad. In addition, it is difficult to accurately control the location and characteristics of the tumor by the induction method. The transplanted tumor model is established by inoculating tumor cells into the body of experimental animals, but the heart is a highly specialized organ, and its establishment of a transplant model faces many technical difficulties. In addition, the biological characteristics of transplanted tumor cells may change due to factors such as immune rejection.

[0004] Therefore, it is necessary to develop a method to construct a mouse model of primary cardiac tumors that does not require induction and will not produce immune rejection. Summary of the invention

[0005] The present invention combines gene conditional knockout technology, breeding, screening and identification technology to develop a method for constructing a primary cardiac tumor mouse model. The construction method does not require physical or chemical induction of experimental mice, avoids the problem of immune rejection, and has a stable model acquisition rate.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides a method for constructing a mouse model of primary cardiac tumor, comprising: (1) conditionally knocking out Scgb1a1; flox / + Mice were bred to express Scgb1a1 flox / flox Homozygous mice were considered F 0.5 (2) F 0.5 Generation with Myh6 Cre+ Scgb1a1 was screened after breeding of tool mice flox / + -Myh6 Cre+ The offspring are considered F 1 Generation; (3) F 1 After generation of breeding, Scgb1a1 was selected flox / flox -Myh6Cre+ The offspring are considered F 2 Generation; (4) F 2 After further breeding, Scgb1a1 was selected flox / flox -Myh6 Cre+ The offspring are considered F 2+1 Generation; F 2+1 The mouse model of primary cardiac tumor was obtained.

[0008] Preferably, the above construction method further comprises: F 2+1 After generation of breeding, Scgb1a1 was selected flox / flox -Myh6 Cre+ The offspring is F 3+1 Generation, and so on, F n After generation of breeding, Scgb1a1 was selected flox / flox -Myh6 Cre+ The offspring is F n+1 Generation; F n+1 The mouse model of primary cardiac tumor was obtained in the first generation; n ≥ 3 and is an integer.

[0009] Preferably, the above F n+1 Substitute F 4 Generation, F 3 F 4 Generation, F 4 The mouse model of primary cardiac tumor was obtained.

[0010] Preferably, the above F n+1 Substitute F 5 Generation, F 4 F 5 Generation, F 5 The mouse model of primary cardiac tumor was obtained.

[0011] Preferably, the above-mentioned Scgb1a1 conditional knockout Scgb1a1 flox / + The mouse construction method includes: conditionally knocking out the second exon Exon 2 and the third exon Exon 3 of the Scgb1a1 gene.

[0012] Another aspect of the present invention further provides a primary cardiac tumor mouse model, which is constructed by the construction method described in the present invention.

[0013] The beneficial effects of the present invention include: the method for constructing a primary cardiac tumor mouse model provided by the present invention is to flox / flox Homozygous mice and Myh6 Cre+ The tool mice were bred from F 3 Starting from the first generation of mice, a mouse model of primary cardiac tumors can be obtained, with a model success rate of 21.15%; 4The modeling ratio of the generation is higher than that of F 3 generation, reaching 40.48%; F 5 The modeling ratio of the generation is higher than that of F 4 The construction method does not require any physical or chemical induction. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the targeting strategy for conditional knockout of Scgb1a1;

[0015] Figure 2 This is the change of ejection fraction in the mouse model of primary cardiac tumor;

[0016] Figure 3 This is the body weight change of the mouse model with primary cardiac tumor;

[0017] Figure 4 Abnormal electrocardiogram in the mouse model of primary cardiac tumor;

[0018] Figure 5 This is the left atrial tumor condition of the mouse model of primary cardiac tumor;

[0019] Figure 6 This is the coronal section of the heart of a mouse with primary cardiac tumor, paraffin section, and HE staining;

[0020] Figure 7 This is a paraffin section of cardiac tumor tissue in a mouse model of primary cardiac tumor, HE staining;

[0021] Figure 8 Paraffin sections of tumor tissues of mouse models of primary cardiac tumors, immunofluorescence staining;

[0022] Fig. 9 Scgb1a1 flox / + -Myh6 Cre+ Breeding diagram. DETAILED DESCRIPTION

[0023] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0024] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0025] The present invention provides a method for constructing a mouse model of primary cardiac tumor, comprising: (1) conditionally knocking out Scgb1a1; flox / + Mice were bred to express Scgb1a1 flox / flox Homozygous mice were considered F 0.5 (2) F 0.5 Generation with Myh6 Cre+ Scgb1a1 was screened after breeding of tool mice flox / + -Myh6 Cre+ The offspring are considered F 1 Generation; (3) F 1 After generation of breeding, Scgb1a1 was selected flox / flox -Myh6 Cre+ The offspring are considered F 2 Generation; (4) F 2 After further breeding, Scgb1a1 was selected flox / flox -Myh6 Cre+ The offspring are considered F 2+1 Generation; F 2+1 The mouse model of primary cardiac tumor was obtained.

[0026] It should be noted that, in the present invention, F 3 The mouse model of primary cardiac tumor can be obtained in the first generation, and the acquisition rate is 21.15%, so it is necessary to breed to F 3 A mouse model of primary cardiac tumors can be obtained in just one generation; in addition, the peak time of tumor occurrence is 4-5 months of age, and the overall time is short.

[0027] In some specific examples, the above construction method further includes: F 2+1 After generation of breeding, Scgb1a1 was selected flox / flox -Myh6 Cre+ The offspring is F 3+1 Generation, and so on, F n After generation of breeding, Scgb1a1 was selected flox / flox -Myh6 Cre+The offspring is F n+1 Generation; F n+1 The mouse model of primary cardiac tumor was obtained in the first generation; n ≥ 3 and is an integer.

[0028] In some specific examples, the above F n+1 Substitute F 4 Generation, F 3 F 4 Generation, F 4 The mouse model of primary cardiac tumor was obtained.

[0029] In some specific examples, the above F n+1 Substitute F 5 Generation, F 4 F 5 Generation, F 5 The mouse model of primary cardiac tumor was obtained.

[0030] It should be noted that the construction method in the present invention is to 3 In the past 20 years, mouse models with primary cardiac tumors have been obtained, and with the continuous cultivation, the acquisition rate of mouse models with primary cardiac tumors has increased significantly, such as F 3 The acquisition rate of primary cardiac tumor mouse models was 21.15%; 4 The acquisition rate of primary cardiac tumor mouse models was 40.48%; 5 The acquisition rate of primary cardiac tumor mouse models can reach 53.33%.

[0031] In some specific examples, the above-mentioned Scgb1a1 conditional knockout Scgb1a1 flox / + The mouse construction method includes: conditionally knocking out the second exon Exon 2 and the third exon Exon 3 of the Scgb1a1 gene.

[0032] An embodiment of the present invention also provides a primary cardiac tumor mouse model, which is constructed by the construction method described in the present invention.

[0033] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0034] In the following example, strain name: C57BL / 6J-Scgb1a1 em1(flox)Cya , gene name: Scgb1a1, mouse strain background: C57BL / 6J, modification method: conditional knockout.

[0035] In the following example, Scgb1a1 conditional knockout mice (Scgb1a1 flox / +Mouse) was commissioned by Saiye (Suzhou) Biotechnology Co., Ltd. to construct the target strategy. Figure 1 As shown, specifically, the second exon (Exon2) and the third exon (Exon3) of the Scgb1a1 gene were conditionally knocked out, that is, two loxP restriction sites were inserted into the 5' end and 3' end of Exon2 and Exon3, respectively, and corresponding PCR identification primers were designed. Figure 1 As shown: the F1 / R1 amplification region is the 5'arm end loxp site, and a 207bp fragment is obtained, and the F2 / R2 amplification region is the 3'arm end loxp site, and a 200bp fragment is obtained. The expansion of both positive bands comprehensively indicates that the targeting is successful.

[0036] In the following example, Myh6 Cre+ The tool mice were also purchased from Saiye (Suzhou) Biotechnology Co., Ltd.

[0037] In the following example, a mouse model of primary cardiac tumors was identified by testing the mouse echocardiogram, body weight, electrocardiogram, left atrial tumor, and pathological sections, as follows:

[0038] (1) Echocardiography in mice

[0039] Cardiac B-ultrasound examination is the main experimental method for identifying and detecting the occurrence of primary cardiac tumors. The cardiac tumors formed in this model are more likely to occur in the left atrium, and occasionally the left ventricle and right atrium are involved. The tumor appears as a solid tumor, with a filling-like growth in the left atrium, which seriously affects the cardiac ejection function. Therefore, in the B-ultrasound examination, the ejection fraction is significantly decreased, and it presents a progressive form, that is, the ejection fraction continues to decrease as the tumor grows; in the middle and late stages, the ejection fraction is less than 10%; if Figure 2 As shown;

[0040] (2) Weight loss

[0041] Tumor cachexia causes mice to develop cardiac tumors and experience a significant decrease in body weight, with a 15%-20% weight loss in one month. Figure 3 As shown;

[0042] (3) Abnormal electrocardiogram

[0043] Cardiac tumors change the electrical conduction of the heart and the normal sinus rhythm. Abnormal electrocardiograms mainly manifest as slow heart rate, high and sharp P waves, paroxysmal atrioventricular block (AVB) and atrial fibrillation (AF) under anesthesia, such as Figure 4 As shown;

[0044] (4) Left atrial aneurysm

[0045] Mice with positive results in the above three tests were anesthetized and the chest was opened. It was found that the heart was dilated like heart failure, the left atrium was enlarged, and a light yellow mass was seen in the left atrium, protruding from the atrial wall. Figure 5 As shown;

[0046] (5) Pathological sections

[0047] Paraffin section, HE staining, clearly visible tumor tissue in the left atrium (see Figure 6 , in which the red circle shows a clear left atrial tumor tissue, 20 times); under the microscope, scattered spindle-shaped and star-shaped tumor cells can be seen in the mucinous matrix (see Figure 7 , where the arrows indicate spindle-shaped and star-shaped tumor cells, 40x microscope); Immunofluorescence examination: Vimentin (+), Calretinin (+), CD31 (+), CD34 (+), Desmin (+), α-SMA (+) (see Figure 8 , 40x lens); specifically including: the heart was sliced ​​using a cryostat to obtain 10 mm thick coronal sections. Immunofluorescence staining was used to detect the expression of vimentin, calretinin, CD31, CD34, desmin and α-SMA. For immunofluorescence, tissue sections were washed three times for 5 minutes each in phosphate-buffered saline containing 0.1% Triton X-100 (PBST). The sections were blocked with 10% goat serum or 10% horse serum in PBST for 1 hour at room temperature and incubated with primary antibodies overnight at 4°C (primary antibody list). The next day, tissue sections were washed three times for 5 minutes each with PBS at room temperature. The tissues were incubated with Alexa Fluor 647 donkey anti-rabbit IgG H&L and Alexa Fluor 647 donkey anti-mouse IgG H&L secondary antibodies for 2 hours in the dark at room temperature. After washing three times in PBS for 5 minutes each, the tissues were mounted, the slides were covered with anti-fading fluorescent mounting medium containing DAPI, and sealed with nail polish. The images were collected by taking photos on an Olympus BX63 fluorescence microscope. The red luminous area is the immunofluorescence positive staining.

[0048] Among them, the list of primary antibodies for immunofluorescence staining is as follows:

[0049] 1) CD34 (E2J1K) Rabbit mAb, Cell Signaling technology (CST), Cat# 26233, dilution ratio 1:200;

[0050] 2) Calretinin (E7R6O) Rabbit mAb, Cell Signaling technology (CST), Cat#92635, dilution ratio 1:100;

[0051] 3) Vimentin (D21H3) Rabbit mAb, Cell Signaling technology (CST), Cat#92635, dilution ratio 1:100;

[0052] 4)α-Smooth Muscle Actin(D4K9N) Rabbit mAb, Cell Signaling Technology (CST), Cat#92635, dilution ratio 1:200;

[0053] 5) Desmin (10H7D2), Santa Cruz Biotechnology, sc-65983, dilution ratio 1:100;

[0054] 6) CD31 (PECAM-1) (D8V9E) Rabbit mAb, Cell Signaling technology (CST), Cat#26233, dilution ratio 1:100;

[0055] List of secondary antibodies for immunofluorescence staining:

[0056] 1) Alexa Fluor 647Donkey Anti-Rabbit IgG (H+L), Abcam, Cat#ab150075;

[0057] 2) Alexa Fluor 647Donkey Anti-Mouse IgG (H+L), Abcam, Cat#ab150107.

[0058] Example 1

[0059] according to Fig. 9 The breeding method shown is as follows:

[0060] (1) Scgb1a1 flox / + Considered as F 0 generation, utilizing Scgb1a1 flox / + Reproduction, obtaining Scgb1a1 flox / flox Homozygous mice were considered F 0.5 generation;

[0061] (2) Using F0.5 Generation with Myh6 Cre+ The mating of tool mice resulted in three different genotypes of mice, among which the genotype was identified as Scgb1a1 flox / + -Myh6 Cre+ The offspring of 1 generation;

[0062] (3) Using F 1 After 4 generations of breeding, 4 different genotypes of mice were obtained, among which the genotype was identified as Scgb1a1 flox / flox -Myh6 Cre+ The offspring of 2 generation;

[0063] (4) F 2 The next generation was further propagated, and the genotype was identified as Scgb1a1 flox / flox -Myh6 Cre+ The offspring of 3 generation;

[0064] (5) F 3 The next generation was further propagated, and the genotype was identified as Scgb1a1 flox / flox -Myh6 Cre+ The offspring of 4 generation.

[0065] The above-mentioned primary cardiac tumor mouse model identification method was used to screen F 3 Generation, F 4 Generation and F 5 The primary cardiac tumor mouse model was established in the previous generation, and the model success rate was calculated. The results are shown in Tables 1, 2 and 3 below.

[0066] In Table 1, Table 2 and Table 3, M = Month, which is the age of the mouse in months; 7M refers to the age of the mouse at 7 months; 6M refers to the age of the mouse at 6 months; and so on; 2 and 3 months are the starting time for statistics, and 52 is the total number of mice involved in the statistics; the total number of mice counted at 3 months of age (52) = the number of mice without tumors (50) + the number of mice with heart tumors (2); and so on: 50 is the total number of mice counted at 4 months of age (50) = the number of mice without tumors (47) + the number of mice with heart tumors (3). According to the above calculation, the total number of mice counted at 5 months of age should be (47), but there are actually only 36; the reason is that: 7 of the mice were treated in other experiments and could no longer be included in the statistics; the same is true for the following 6 and 7 months of age.

[0067] Table 1F 3 The rate of success

[0068]

[0069] From Table 1 above, we can see that F 3 The total success rate of the first generation reached 21.15%, and the weighted average success rate was 5.93%.

[0070] Table 2F 4 The rate of success

[0071]

[0072] From Table 2 above, we can see that F 4 The total mold success rate reached 41.46%, and the weighted average mold success rate was 24.22%.

[0073] Table 3F 5 The rate of success

[0074]

[0075] From Table 3 above, we can see that F 5 The success rate of the assembly reached 53.33%.

[0076] Comparing the data in Table 1, Table 2 and Table 3, we can see that F 4 The generation rate is higher than F 3 Generation modulus, F 5 The success rate of the first generation is higher than that of the F4 generation, indicating that with the continuous reproduction of pure knockout mice, the knockout efficiency continues to increase, and the success rate of tumor mice is getting higher and higher.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A method for constructing a primary cardiac tumor mouse model, characterized in that: include: (1) Conditional knockout of Scgb1a1 flox / + Mice were bred to express Scgb1a1 flox / flox Homozygous mice were considered F 0.5 (2) F 0.5 Generation with Myh6 Cre+ Scgb1a1 was screened after breeding of tool mice flox / + -Myh6 Cre+ The offspring of the F1 generation were considered as F1 generation; (3) After the F1 generation was propagated, Scgb1a1 was screened flox / flox -Myh6 Cre+ The offspring of the F2 generation were considered as F2 generation; (4) The F2 generation was further propagated and Scgb1a1 was screened out. flox / flox -Myh6 Cre+ The offspring are considered F 2+1 Generation; F 2+1 The mouse model of primary cardiac tumor was obtained.

2. The method for constructing a primary cardiac tumor mouse model according to claim 1, characterized in that: The construction method also includes: F 2+1 After generation of breeding, Scgb1a1 was selected flox / flox -Myh6 Cre+ The offspring is F 3+1 Generation, and so on, F n After generation of breeding, Scgb1a1 was selected flox / flox -Myh6 Cre+ The offspring is F n+1 generation; F n+1 The mouse model of primary cardiac tumor was obtained in the first generation; n ≥ 3 and is an integer.

3. The construction method according to claim 2, characterized in that: F n+1 The F3 generation was bred into the F4 generation, and the F4 generation obtained a primary cardiac tumor mouse model.

4. The construction method according to claim 3, characterized in that: F n+1 The F5 generation was bred from the F4 generation to obtain the F5 generation, and the F5 generation obtained a primary cardiac tumor mouse model.

5. The construction method according to any one of claims 1 to 4, characterized in that: Conditional knockout of Scgb1a1 flox / + The mouse construction method includes: conditionally knocking out the second exon Exon 2 and the third exon Exon 3 of the Scgb1a1 gene.

6. A primary cardiac tumor mouse model, characterized in that: The method is constructed by any one of claims 1 to 5.

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