A method for constructing an in-situ pulmonary nodule mouse model

Abstract

The present invention discloses a method for constructing an in-situ pulmonary nodule mouse model, which comprises the following steps: 1) Hybridize loxp-stop-loxp KrasG12D transgenic mice with loxp-Tgfbr2-loxp transgenic mice; 2) Intratracheally instill a low dose of adenovirus (AdCre) with the activity of an enzyme that recognizes specific gene sequence cleavage into the F0 hybrid mice obtained in step 1) or their offspring. The present invention also discloses the application of the constructed in-situ pulmonary nodule mouse model in the research on the pathogenesis of in-situ pulmonary nodule diseases and the screening of new drugs for in-situ pulmonary nodules.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for constructing an in-situ pulmonary nodule mouse model. Background Art

[0002] A pulmonary nodule is defined as a round, focal, density-increased solid or sub-solid pulmonary abnormal shadow with a diameter less than 3 cm in the lung detected by conventional chest CT plain scan or low-dose chest CT screening. Currently, in addition to early surgical treatment, there is no drug treatment method for pulmonary nodules. Regular CT follow-up will inevitably bring radiation exposure. At the same time, the invasive method of pulmonary nodule puncture biopsy used clinically will also bring some unnecessary postoperative complications. In some recent studies, it has been found that a part of malignant pulmonary nodules with an indolent biological behavior have the characteristic of slow imaging progression, especially common in a type of pulmonary nodules with ground-glass imaging, which adds difficulty to the clinical imaging differential diagnosis of pulmonary nodules.

[0003] Recently, through a comparative study of large-scale retrospective clinical samples, it has been found that there is an up-regulation of the expression of a group of pro-cancer genes and a down-regulation of the expression of another group of anti-cancer genes in malignant pulmonary nodules. The former includes the Kras gene, and the latter includes genes such as P53 and Tgfbr2. Currently, internationally, by the method of airway instillation of adenovirus (AdCre) with the activity of a specific gene sequence recognition and cleavage enzyme, the gene editing function of respiratory epithelial cells at specific sites can be achieved, such as the KrasG12DP53- / -(KP) malignant lung cancer mouse model constructed by Tylor Jacks.

[0004] In summary, it is of great significance for the prognosis of pulmonary nodule patients to develop a suitable mouse model for the early differential diagnosis of benign and malignant pulmonary nodules, explore the molecular mechanism of the transformation between benign and malignant pulmonary nodules, and screen effective treatment means for specific targets. Summary of the Invention

[0005] To solve the above problems, the present invention provides a method for constructing an in-situ pulmonary nodule mouse model based on genetic engineering technology. The constructed mouse model can be used for the study of the pathogenesis of the transformation between benign and malignant pulmonary nodules and the screening of new drugs.

[0006] Specifically, in the first aspect of the present invention, a method for constructing an in-situ pulmonary nodule mouse model is provided, including the following steps: 1) Hybridize loxp-stop-loxp KrasG12D transgenic mice with loxp-Tgfbr2-loxp transgenic mice; 2) Perform airway instillation of adenovirus (AdCre) with the activity of a specific gene sequence recognition and cleavage enzyme on the F0 hybrid mice obtained in step 1) or their offspring.

[0007] In some embodiments, the use concentration of the adenovirus (AdCre) is 10 6 -10 8 pfu; more preferably, the use concentration of the adenovirus (AdCre) is 10 7 -5×10 7 pfu.

[0008] In some embodiments, the use concentration of the adenovirus (AdCre) is 2.5×10 7 pfu.

[0009] In some embodiments, before step 1), the loxp-stop-loxp KrasG12D transgenic mice are verified using the primers of SEQ ID NO: 1-2 or SEQ ID NO: 3-4.

[0010] In some embodiments, before step 1), the loxp-Tgfbr2-loxp transgenic mice are verified using the primers of SEQ ID NO: 5-8.

[0011] In some embodiments, after step 2), the mutation of the loxp-stop-loxp KrasG12D locus of the modeled mice is verified using SEQ ID NO: 9-12;

[0012] In some embodiments, after step 2), the deletion of the loxp-Tgfbr2-loxp locus of the modeled mice is verified using the primer SEQ ID NO: 13-14.

[0013] The second aspect of the present invention provides the application of the mouse model obtained by the method according to the first aspect of the present invention in the study of the pathogenesis of in-situ lung nodule diseases.

[0014] The second aspect of the present invention provides the application of the mouse model according to the first aspect of the present invention in the screening of new drugs for in-situ lung nodules.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] 1) The present invention induces gene mutations in mouse in-situ lung epithelial cells by low-dose airway instillation of adenovirus, forms in-situ somatic carcinogenesis, and establishes the first in-situ lung nodule animal model. Compared with the KP model and the lung nodule mouse KrasG12DTgfbr2- / -(KT) model with high-dose (10 9 pfu) KrasG12D mutation combined with Tgfbr2 deletion, low-dose (2.5*10 7The pfu)KT model is characterized by a slower progression of the lesion, providing a good observation time window for observing the process of the transformation of pulmonary nodules from the benign stage to the malignant stage, especially the pathophysiological changes in the microenvironment of early pulmonary nodules.

[0017] 2) The growth pattern of the in-situ pulmonary nodules of the present invention highly fits the biological behavior of human pulmonary nodules.

[0018] 3) Compared with the nude mouse xenograft tumor model, it completely retains the normal immune function of the mouse and is more suitable for studying the pathogenesis of the tumor microenvironment of in-situ lung adenocarcinoma.

[0019] 4) It provides an ideal animal model for the research and development of the pathogenesis of in-situ pulmonary nodule diseases and new drug screening, filling the gap in such animal models. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] By reading the detailed description of the non-limiting embodiments with reference to the following drawings, other features, objects, and advantages of the present invention will become more apparent:

[0021] Figure 1 Schematic diagram showing the CT changes of the low-dose KT mice of the present invention.

[0022] Figure 2 Showing the effects of different mutant mouse models (KP model, KT model) and different doses of adenovirus infusion on the KT model. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings of the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0024] Unless otherwise defined, the technical terms or scientific terms used herein shall have the ordinary meanings understood by those of ordinary skill in the art to which the present invention belongs.

[0025] Example 1 Construction of the KT Mouse Model

[0026] By crossing transgenic mice [1] with the loxp-stop-loxp KrasG12D locus and transgenic mice [2] with the loxp-Tgfbr2-loxp locus, F0 generation mice were obtained;

[0027] Extract the tail DNA of F0 generation mice, perform PCR amplification and send the products for sequencing. The primer information for PCR amplification is shown in Table 1. Transgenic mice with the loxp-stop-loxp KrasG12D locus were verified using primer numbers 1 and 2, and transgenic mice with the loxp-Tgfbr2-loxp locus were verified using primer number 3.

[0028] Table 1 Primer Information for PCR

[0029]

[0030] Table 2 PCR Reaction System

[0031] Reaction Components Volume(ul) ddH2O 16 2X Taq Plus Master Mix(Dye Plus) 25 Cleavage products 5 Primer 1 (10uM) 2 Primer 2 (10uM) 2

[0032] Table 3 PCR Program

[0033] Seg. Temp. Time Cycle 1 94℃ 5min 2 94℃ 30s 3 55℃ 30s 4 72℃ 30s 2-4,35 cycles 5 72℃ 7min

[0034] When the mice were 8 - 10 weeks old and male mice weighed over 25 g, an in-situ malignant lung nodule mouse model was constructed by intratracheal instillation of adenovirus (AdCre) with the activity of an enzyme that recognizes specific gene sequence cleavage. Among them, the adenovirus was set at a low concentration group of 2.5*10 7 pfu and a high concentration group of 10 9 pfu.

[0035] Extract the DNA of the fresh lung tissue of the mice after modeling, perform PCR amplification and send the products for sequencing. The primer information for amplification is shown in Table 1. Primer numbers 4 and 5 were used to verify the mutation of the loxp-stop-loxp KrasG12D locus, and primer number 6 was used to verify the deletion of the loxp-Tgfbr2-loxp locus.

[0036] The PCR reaction system and PCR program are shown in Table 2 and Table 3 respectively.

[0037] Figure 1 Show the modeling results of the 2.5*10 7 pfu group of adenovirus. CT images of KT mice at 4 weeks, 6 weeks, 9 weeks, 11 weeks, 13 weeks, and 15 weeks were selected respectively. It was found that in-situ lung nodules appeared in the mice starting from 4 weeks, and as the modeling time extended, the lung nodules changed from the benign stage to the malignant stage.

[0038] Example 2 Functional Verification of KT Mouse Model

[0039] Further explored the effects of different mutant mouse models (KP model, KT model) and different doses of adenovirus instillation on the KT model. The results showed that compared with the KP model and the high dose (10 9Compared with the pfu adenovirus) KT model [3], the KrasG12D mutation combined with Tgfbr2 deletion in the low-dose (2.5*10 7 pfu adenovirus) KrasG12D Tgfbr2- / -(KT) mouse model of lung nodules has the characteristic of slower lesion progression, providing a good observation time window for observing the process of lung nodules changing from the benign stage to the malignant stage, especially the pathophysiological changes in the microenvironment of early lung nodules ( Figure 2 ).

[0040] References:

[0041] 1. Jackson, E.L., et al., Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev, 2001. 15(24): p. 3243-8.

[0042] 2. Chytil, A., et al., Conditional inactivation of the TGF-beta type II receptor using Cre:Lox. Genesis, 2002. 32(2): p. 73-5.

[0043] 3. Borczuk, A.C., et al., Progression of human bronchioloalveolar carcinoma to invasive adenocarcinoma is modeled in a transgenic mouse model of K-ras-induced lung cancer by loss of the TGF-β type II receptor. Cancer Res, 2011. 71(21): p. 6665-75.

[0044] The foregoing has shown and described the basic principles, main features and advantages of the present invention. For a person skilled in the art, it is obvious that the present invention is not limited to the details of the above-mentioned exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, it is intended to embrace all changes that fall within the meaning and scope of the equivalent elements of the claims in the present invention. Any reference signs in the claims should not be construed as limiting the claims concerned.

[0045] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. A person skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by a person skilled in the art.

Claims

1. A method for constructing an in-situ pulmonary nodule mouse model, characterized in that, It includes the following steps: 1) Hybridize loxp-stop-loxp KrasG12D transgenic mice with loxp-Tgfbr2-loxp transgenic mice; 2) Intratracheally instill adenovirus AdCre with the activity of an enzyme that recognizes a specific gene sequence cleavage into the F0 hybrid mice obtained in step 1) or their offspring.

2. The method according to claim 1, wherein The use concentration of the adenovirus AdCre is lower than 10 9 pfu.

3. According to the method described in claim 2, the use concentration of the adenovirus AdCre is 10 6 -10 8 pfu.

4. According to the method described in claim 3, the use concentration of the adenovirus AdCre is 10 7 -5×10 7 pfu.

5. The method according to claim 4, wherein The use concentration of the adenovirus AdCre is 2.5×10 7 pfu.

6. The method according to claim 1, characterized in that, Before step 1), verify the loxp-stop-loxp KrasG12D transgenic mice using the primers of SEQ ID NO: 1-2 or SEQ ID NO: 3-4.

7. The method according to claim 1, characterized in that, Before step 1), verify the loxp-Tgfbr2-loxp transgenic mice using the primers of SEQ ID NO: 5-8.

8. The method according to claim 1, characterized in that, After step 2), verify the mutation at the loxp-stop-loxp KrasG12D locus of the modeled mice using SEQ ID NO: 9-12.

9. The method according to claim 1, characterized in that, After step 2), verify the deletion of the loxp-Tgfbr2-loxp locus of the modeled mice using the primers SEQ ID NO: 13-14.

10. Application of the mouse model obtained by the method according to any one of claims 1-9 in the study of the pathogenesis of in situ pulmonary nodule diseases.

11. Application of the mouse model obtained by the method according to any one of claims 1-9 in the screening of new drugs for in situ pulmonary nodules.

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