A method for constructing a mouse model of primary cardiac tumors

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to a method for constructing a mouse model of primary cardiac tumor. The method includes: (1) Breeding Scgb1a1 conditional knockout Scgb1a1 flox / + mice to obtain Scgb1a1 flox / flox homozygous mice, regarded as the F 0.5 generation; (2) Breeding the F 0.5 generation with Myh6 Cre+ tool mice, and screening the offspring of Scgb1a1 flox / + -Myh6 Cre+ as the F1 generation; (3) Breeding the F1 generation and screening the offspring of Scgb1a1 flox / flox -Myh6 Cre+ as the F2 generation; (4) Continuing to breed the F2 generation and screening the offspring of Scgb1a1 flox / flox -Myh6 Cre+ as the F 2+1 generation; The F 2+1 generation obtains a mouse model of primary cardiac tumor. This construction method does not require any physical and chemical induction of mice.

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 primary cardiac tumor. Background Art

[0002] Mouse tumor models play an important role in oncology research. The characteristics of being easily obtained, having a short growth cycle, and being easy to operate make mice the commonly used tumor model animals. Through mouse tumor models, the growth process of human tumors can be simulated, providing an important experimental platform for studying the occurrence, development, and treatment of tumors.

[0003] Currently, the methods for constructing tumor models mainly include spontaneous or induced tumor models, which mainly rely on the tumor incidence rate of experimental animals themselves or the administration of carcinogens to induce tumors. The spontaneous rate of primary cardiac tumors is extremely low, and there is no report of spontaneous primary cardiac tumors in adult mice at home and abroad; moreover, the induced method is difficult to precisely control the occurrence location and characteristics of tumors; the transplanted tumor model is established by inoculating tumor cells into experimental animals, but as a highly specialized organ, the establishment of its transplantation model faces many technical difficulties; and the transplanted tumor cells may change their biological characteristics due to factors such as immune rejection.

[0004] Therefore, it is necessary to develop a method for constructing a mouse model of primary cardiac tumor that does not require induction and does 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 mouse model of primary cardiac tumor. This 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] To achieve the above object, the present invention can adopt the following technical solutions:

[0007] On the one hand, the present invention provides a method for constructing a mouse model of primary cardiac tumor, including: (1) Breeding Scgb1a1 conditional knockout Scgb1a1 flox / + mice to obtain Scgb1a1 flox / flox homozygous mice regarded as the F 0.5 generation; (2) Breeding the F 0.5 generation with Myh6 Cre+ tool mice and then screening out the offspring of Scgb1a1 flox / + -Myh6 Cre+ as the F1 generation; (3) After breeding the F1 generation, screening out Scgb1a1 flox / flox -Myh6 Cre+The offspring are regarded as the F2 generation; (4) After continuous breeding of the F2 generation, Scgb1a1 is screened out. flox / flox -Myh6 Cre+ The offspring are regarded as F 2+1 generation; F 2+1 generation to obtain a mouse model of primary cardiac tumor.

[0008] Preferably, the above construction method further includes: After breeding of the F 2+1 generation, Scgb1a1 is screened out. flox / flox -Myh6 Cre+ The offspring are the F 3+1 generation, and so on. After breeding of the F n generation, Scgb1a1 is screened out. flox / flox -Myh6 Cre+ The offspring are the F n+1 generation; The F n+1 generation to obtain a mouse model of primary cardiac tumor; n≥3 and is an integer.

[0009] Preferably, the above F n+1 generation is the F4 generation. Breed the F3 generation to obtain the F4 generation, and the F4 generation to obtain a mouse model of primary cardiac tumor.

[0010] Preferably, the above F n+1 generation is the F5 generation. Breed the F4 generation to obtain the F5 generation, and the F5 generation to obtain a mouse model of primary cardiac tumor.

[0011] Preferably, the construction method of the above Scgb1a1 conditional knockout of Scgb1a1 flox / + mice includes: Conditionally knocking out exon 2 and exon 3 of the Scgb1a1 gene.

[0012] On the other hand, the present invention also provides a mouse model of primary cardiac tumor, which is constructed by the construction method described in the present invention.

[0013] The beneficial effects of the present invention include: The construction method of the mouse model of primary cardiac tumor provided by the present invention is obtained by breeding Scgb1a1 flox / flox homozygous mice and Myh6 Cre+ tool mice. Starting from the F3 generation of mice, a mouse model of primary cardiac tumor can be obtained, and the modeling rate is 21.15%; The modeling ratio of the F4 generation is higher than that of the F3 generation, reaching 40.48%; The modeling ratio of the F5 generation is higher than that of the F4 generation and can reach 53.33%. This construction method does not require any physical and chemical induction. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic diagram of the Scgb1a1 conditional knockout targeting strategy;

[0015] Figure 2 Ejection fraction changes in a mouse model of primary cardiac tumor

[0016] Figure 3 Body weight changes in a mouse model of primary cardiac tumor

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

[0018] Figure 5 Left atrial tumor mass in a mouse model of primary cardiac tumor

[0019] Figure 6 Coronal section of the heart of a mouse with primary cardiac tumor, paraffin section, HE staining

[0020] Figure 7 Paraffin section of the cardiac tumor tissue of a mouse with primary cardiac tumor, HE staining

[0021] Figure 8 Paraffin section of the tumor tissue of a mouse with primary cardiac tumor, immunofluorescence staining

[0022] Figure 9 For Scgb1a1 flox / + -Myh6 Cre+ Breeding schematic diagram Detailed implementation manners

[0023] The examples given are for better illustration of the present invention, but the content of the present invention is not limited only to the examples given. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation manners according to the above invention content still fall within the protection scope of the present invention.

[0024] The terms used herein are only for describing specific embodiments and are not intended to limit the present disclosure. Unless having an apparently different meaning in the context, the expressions in the singular form include the plural form. As used herein, it should be understood that terms such as "including", "having", "containing" are intended to indicate the existence 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 their combinations may exist or can be added. As used herein, depending on the context, " / " can be interpreted as "and" or "or".

[0025] An embodiment of the present invention provides a method for constructing a mouse model of primary cardiac tumor, including: (1) Conditionally knocking out Scgb1a1 in Scgb1a1 flox / + mice for breeding to obtain Scgb1a1flox / flox Homozygous mice are regarded as the F 0.5 generation; (2) After breeding the F 0.5 generation with Myh6 Cre+ tool mice and screening, the offspring of Scgb1a1 flox / + -Myh6 Cre+ are regarded as the F1 generation; (3) After breeding the F1 generation and screening, the offspring of Scgb1a1 flox / flox -Myh6 Cre+ are regarded as the F2 generation; (4) After continuous breeding of the F2 generation and screening, the offspring of Scgb1a1 flox / flox -Myh6 Cre+ are regarded as the F 2+1 generation; The F 2+1 generation obtains a mouse model of primary heart tumor.

[0026] It should be noted that in the present invention, a mouse model of primary heart tumor can be obtained in the F3 generation, and the acquisition rate is 21.15%. Therefore, a mouse model of primary heart tumor can be obtained by breeding to the F3 generation; in addition, the peak time of tumor occurrence is at 4-5 months of age, and the overall time is short.

[0027] In some specific examples, the above construction method further includes: After breeding the F 2+1 generation and screening, the offspring of Scgb1a1 flox / flox -Myh6 Cre+ are the F 3+1 generation, and so on. After breeding the F n generation and screening, the offspring of Scgb1a1 flox / flox -Myh6 Cre+ are the F n+1 generation; The F n+1 generation obtains a mouse model of primary heart tumor; n≥3 and is an integer.

[0028] In some specific examples, the above F n+1 generation is the F4 generation. Breed the F3 generation to obtain the F4 generation, and the F4 generation obtains a mouse model of primary heart tumor.

[0029] In some specific examples, the above F n+1 generation is the F5 generation. Breed the F4 generation to obtain the F5 generation, and the F5 generation obtains a mouse model of primary heart tumor.

[0030] It should be noted that in the construction method of the present invention, a mouse model of primary heart tumor can be obtained from the F3 generation, and with continuous cultivation, the acquisition rate of the mouse model of primary heart tumor increases significantly. For example, the acquisition rate of the mouse model of primary heart tumor in the F3 generation is 21.15%; while the acquisition rate of the mouse model of primary heart tumor in the F4 generation is 40.48%; the acquisition rate of the mouse model of primary heart tumor in the F5 generation can reach 53.33%.

[0031] In some specific examples, the method for constructing the above Scgb1a1 conditional knockout Scgb1a1 flox / + mice includes: conditionally knocking out Exon 2 of the Scgb1a1 gene and Exon 3 of the Scgb1a1 gene.

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

[0033] To better understand the present invention, the content of the present invention will be further clarified below with specific examples, but the content of the present invention is not limited to the following examples.

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

[0035] In the following examples, the Scgb1a1 conditional knockout mice (Scgb1a1 flox / + mice) were constructed by Cyagen Biosciences (Suzhou) Inc., among which the targeting strategy is as Figure 1 shown. Specifically, Exon 2 (Exon2) and Exon 3 (Exon3) of the Scgb1a1 gene were conditionally knocked out, that is, two loxP cleavage sites were inserted into the 5' end and 3' end of Exon2 and Exon3 respectively, and corresponding PCR identification primers were designed. As Figure 1 shown: The amplification region of F1 / R1 is the loxP site at the 5' arm end, obtaining a 207bp fragment, and the amplification region of F2 / R2 is the loxP site at the 3' arm end, obtaining a 200bp fragment. The successful targeting is comprehensively indicated by the amplification of both positive bands.

[0036] In the following examples, the Myh6 Cre+ tool mice were also purchased from Cyagen Biosciences (Suzhou) Inc.

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

[0038] (1) Mouse echocardiogram detection

[0039] Echocardiography is the main experimental method for the identification and detection of primary cardiac tumors; the cardiac tumors formed by this model are prone to occur in the left atrium, and occasionally the left ventricle and right atrium are involved; the tumor presents as a solid tumor, growing in a filling-like manner in the left atrium, seriously affecting the cardiac ejection function. Therefore, in echocardiography, it is manifested as a significant decrease in the ejection fraction, and it shows a progressive form, that is, the ejection fraction continuously decreases as the tumor mass increases; in the middle and late stages, the ejection fraction is less than 10%; as Figure 2 shown;

[0040] (2) Weight loss

[0041] Tumor cachexia leads to a significant decrease in the body weight of mice suffering from cardiac tumors, and the body weight can be reduced by 15%-20% in one month; as Figure 3 shown;

[0042] (3) Abnormal electrocardiogram

[0043] Cardiac tumors change the cardiac electrical conduction and normal sinus rhythm. The main manifestations of abnormal electrocardiogram are slow heart rate, tall and pointed P wave, paroxysmal atrioventricular block (AVB) and atrial fibrillation (AF) under anesthesia, as Figure 4 shown;

[0044] (4) Left atrial tumor mass

[0045] Anesthetize and perform thoracotomy on the mice with positive results in the above 3 tests; it can be seen that the heart shows heart failure-like dilation, the volume of the left atrium increases, and a light yellow mass can be seen in the left atrium, protruding from the atrial wall, as Figure 5 shown;

[0046] (5) Pathological section

[0047] Paraffin section, HE staining, it can be clearly seen that there is tumor tissue in the left atrium (see Figure 6 , in which, a clear left atrial tumor tissue body can be seen within the red circle, 20 times magnification); under the microscope, scattered spindle-shaped and stellate tumor cells can be seen in the mucinous matrix (see Figure 7 , in which, the arrow indicates spindle-shaped and stellate tumor cells, 40 times magnification); immunofluorescence examination: Vimentin(+), Calretinin(+), CD31(+), CD34(+), Desmin(+), α-SMA(+)(see Figure 8, 40x objective lens); specifically, it includes: using a cryostat to section the heart to obtain 10-mm-thick coronary sections. Immunofluorescence staining was used to detect the expression of Vimentin, Calretinin, CD31, CD34, Desmin, and α-SMA. For immunofluorescence, tissue sections were washed 3 times in phosphate-buffered saline containing 0.1% Triton X-100 (PBST) for 5 minutes each time. Sections were blocked with 10% goat serum or 10% horse serum in PBST at room temperature for 1 hour and incubated with primary antibodies overnight at 4°C (list of primary antibodies). The next day, tissue sections were washed 3 times in PBS at room temperature for 5 minutes each time. At room temperature, the tissue was incubated with Alexa Fluor 647 donkey anti-rabbit IgG H&L and Alexa Fluor 647 donkey anti-mouse IgG H&L secondary antibodies in the dark for 2 hours. After washing 3 times in PBS for 5 minutes each time, the tissue was mounted, covered with a coverslip using a fluorescence mounting medium containing DAPI, and sealed with nail polish. Images were captured by photographing on an Olympus BX63 fluorescence microscope. The red-emitting area is the immunofluorescence positive staining.

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

[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 647 Donkey Anti-Rabbit IgG(H+L), Abcam, Cat#ab150075;

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

[0058] Example 1

[0059] According to Figure 9 the breeding method shown below:

[0060] (1) Consider Scgb1a1 flox / + as the F0 generation, and use Scgb1a1 flox / + to breed. The Scgb1a1 flox / flox homozygous mice are regarded as the F 0.5 generation;

[0061] (2) Mate the F 0.5 generation with Myh6 Cre+ tool mice to obtain 3 different genotypes of mice. Among them, the offspring with the genotype identified as Scgb1a1 flox / + -Myh6 Cre+ are regarded as the F1 generation;

[0062] (3) Use the F1 generation to continue breeding to obtain 4 different genotypes of mice. Among them, the offspring with the genotype identified as Scgb1a1 flox / flox -Myh6 Cre+ are regarded as the F2 generation;

[0063] (4) Continue breeding the F2 generation. Among them, the offspring with the genotype identified as Scgb1a1 flox / flox -Myh6 Cre+ are regarded as the F3 generation;

[0064] (5) Continue breeding the F3 generation. Among them, the offspring with the genotype identified as Scgb1a1 flox / flox -Myh6 Cre+ are regarded as the F4 generation.

[0065] Using the above identification method for the mouse model of primary cardiac tumors, the mouse models of primary cardiac tumors in the F3, F4, and F5 generations were screened respectively, and the modeling success rate was calculated. The results are shown in Tables 1, 2, and 3 below.

[0066] In Tables 1, 2, and 3, M = Month, which represents the age of the mice in months; 7M means the mice are 7 months old; 6M means the mice are 6 months old, and so on; 2 and 3 months are the starting times for statistics, and 52 is the total number of mice participating in all statistics; the total number of all mice (52) at 3 months = the number of mice without tumors (50) + the number of mice with cardiac tumors (2); and so on: 50 is the total number of all mice (50) at 4 months = the number of mice without tumors (47) + the number of mice with cardiac tumors (3). According to the above calculation, the total number of all mice at 5 months should be (47), but actually it is only 36; the reason is that 7 of the mice were processed in other experiments and could no longer be included in the statistics; the same is true for 6 and 7 months later.

[0067] Table 1 The situation of the modeling success rate in the F3 generation

[0068]

[0069] As can be seen from Table 1 above, the overall modeling success rate in the F3 generation reached 21.15%, and the weighted average modeling success rate was 5.93%.

[0070] Table 2 The situation of the modeling success rate in the F4 generation

[0071]

[0072] As can be seen from Table 2 above, the overall modeling success rate in the F4 generation reached 41.46%, and the weighted average modeling success rate was 24.22%.

[0073] Table 3 The situation of the modeling success rate in the F5 generation

[0074]

[0075] As can be seen from Table 3 above, the overall modeling success rate in the F5 generation reached 53.33%.

[0076] By comparing the data in Tables 1, 2, and 3, it can be known that the modeling success rate in the F4 generation is higher than that in the F3 generation, and the modeling success rate in the F5 generation is higher than that in the F4 generation. This shows that with the continuous reproduction of homozygous knockout mice, the knockout efficiency is continuously increasing, and the modeling 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 solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A method for constructing a mouse model of primary cardiac tumors, characterized in that, Include: (1) Conditionally knockout Scgb1a1 in Scgb1a1 flox / + mice and breed them to obtain Scgb1a1 flox / flox homozygous mice, regarded as F 0.5 generation; (2) Breed the F 0.5 generation with Myh6 Cre+ tool mice and screen for Scgb1a1 flox / + -Myh6 Cre+ offspring, regarded as F1 generation; (3) Breed the F1 generation and screen for Scgb1a1 flox / flox -Myh6 Cre+ offspring, regarded as F2 generation; (4) Continue to breed the F2 generation and screen for Scgb1a1 flox / flox -Myh6 Cre+ offspring, regarded as F 2+1 generation; The F 2+1 generation yields a mouse model of primary cardiac tumors.

2. The method for constructing a mouse model of primary cardiac tumor according to claim 1, wherein The construction method further includes: F 2+1 After the F generation of reproduction, the offspring of Scgb1a1 flox / flox -Myh6 Cre+ are the F 3+1 generation, and so on. After the F n generation of reproduction, the offspring of Scgb1a1 flox / flox -Myh6 Cre+ are the F n+1 generation; The F n+1 generation obtains a mouse model of primary cardiac tumor; n≥3 and is an integer.

3. The construction method according to claim 2, characterized in that, F n+1 The F generation is the F4 generation. The F3 generation is bred to obtain the F4 generation, and a mouse model with primary heart tumors is obtained from the F4 generation.

4. The construction method according to claim 3, characterized in that, F n+1 It is the F5 generation. The F4 generation is propagated to obtain the F5 generation, and a mouse model of primary heart tumor is obtained from the F5 generation.

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

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