Construction method and application of a sypl1 gene knockout colorectal cancer mouse model

Abstract

The application relates to the technical field of animal model construction, and particularly discloses a Sypl1 gene knockout colorectal cancer mouse model construction method and application, which comprises the following steps: synthesizing a specific target site gRNA of a Sypl1 gene, mixing Cas9 protein and the target site gRNA to obtain an injection compound, microinjecting the injection compound into mouse zygotes, and transplanting the surviving zygotes into the oviducts of pseudopregnant female mice to obtain F0 generation mice, and the mice born after 20 days are Sypl1 gene knockout mouse animal models; and a chemical induction modeling method is used to construct a colorectal cancer model in wild type mice and gene knockout mice. The Sypl1 gene knockout mouse is constructed by using a CRISPR / Cas mediated genome engineering technology Sypl1 combined with the most widely used colorectal cancer chemical inducer azoxymethane / dextran sodium sulfate, a new colorectal cancer mouse model is constructed, compared with a traditional chemical induction model, the tumor formation rate can be significantly improved, and the tumor formation time can be shortened, and a more efficient animal model is provided for colorectal cancer research.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically a method and application for constructing a Sypl1 gene knockout colorectal cancer mouse model. Background Technology

[0002] Colorectal cancer is a malignant tumor that seriously threatens human health. It is the third most common cancer worldwide, with approximately 1.93 million new cases diagnosed globally in 2022. Simultaneously, colorectal cancer is the second leading cause of cancer death worldwide (accounting for 9.3% of all cancer deaths), with approximately 900,000 people dying from it in 2022. Therefore, early diagnosis, new drug development, and exploration of the pathogenesis of colorectal cancer are of great significance for its prevention and treatment.

[0003] Animal models play a crucial role in prevention, treatment, basic research, and preclinical studies. A wide variety of animal models exist for colorectal cancer, with mouse models being the most widely used. Based on the modeling method, they can be categorized into xenograft models, chemically induced models, and transgenic models. Xenograft models are simple to operate, have a high tumor formation rate, and a short tumor formation cycle (3 to 4 weeks), but are not suitable for early-stage colorectal cancer research. Chemically induced models most closely resemble human colorectal cancer in their developmental stages, but the modeling cycle is longer, generally requiring about 3 months. Transgenic models are etiologically close to the natural occurrence of colorectal cancer, but have a lower tumor formation rate and lack invasiveness and metastasis. Therefore, currently used mouse methods for colorectal cancer modeling have several shortcomings, making the development of new modeling methods of great significance. Summary of the Invention

[0004] The purpose of this invention is to address the problems existing in the background art by providing a method for constructing a Sypl1 gene knockout colorectal cancer mouse model.

[0005] To achieve the above objectives, the technical solution adopted by this invention is: a method for constructing a Sypl1 gene knockout colorectal cancer mouse model, comprising the following steps:

[0006] (1) Synthesize a gRNA that is a specific target site of the Sypl1 gene; the gRNA includes an anti-strand sequence and a forward sequence; the nucleotide sequence of the anti-strand sequence is shown in SEQ ID No. 1, and the nucleotide sequence of the forward sequence is shown in SEQ ID No. 2;

[0007] (2) Microinjection: The Cas9 protein, the reverse strand sequence of gRNA and the positive strand sequence of gRNA are mixed to obtain an injection complex, which is microinjected into mouse zygotes. The zygotes are cultured in vitro for 1-2 hours, and the surviving zygotes are transplanted into the oviduct of pseudopregnant mice. Mice born 20 days later are F0 generation mice, which are the Sypl1 gene knockout mouse animal model.

[0008] (3) Colorectal cancer models were constructed in wild-type mice and gene knockout mice using chemical induction modeling methods.

[0009] Furthermore, the target site gRNA targets exon 3 of the Sypl1 gene.

[0010] Furthermore, the PCR identification primers for F0 generation mice include PCR primer 1 and PCR primer 2. The nucleotide sequence of the upstream primer of PCR primer 1 is shown in SEQ ID No. 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 4. The nucleotide sequence of the upstream primer of PCR primer 2 is shown in SEQ ID No. 5, and the nucleotide sequence of the downstream primer of primer 2 is shown in SEQ ID No. 6.

[0011] Furthermore, the wild-type mouse is a wild-type C57BL / 6N mouse.

[0012] The beneficial technical effects of this invention are: this invention constructs a genome using CRISPR / Cas-mediated genome engineering technology. Sypl1 A novel colorectal cancer mouse model was constructed by combining gene knockout mice with azomethane (AOM) / dextrose sulfate (DSS), the most widely used chemical inducer for colorectal cancer. Compared with traditional chemically induced models, this model can significantly improve the tumor formation rate and shorten the tumor formation time, providing a more efficient animal model for colorectal cancer research. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 Example 1: Mice Sypl1 Gene knockout strategy.

[0015] Figure 2 Sequencing verification Sypl1 Gene knockout was successful.

[0016] Figure 3 PCR was used to identify the genotypes of bred young mice.

[0017] Figure 4 Chemically induced colorectal cancer in mice: (A) Modeling method; (B) Changes in body weight during the modeling period.

[0018] Figure 5Changes in colorectal length in mice after colorectal cancer modeling.

[0019] Figure 6 WT mice after colorectal cancer modeling and Sypl1 - / - Volume and number of colorectal tumors in mice.

[0020] Figure 7 Intestinal tissue lesions in mice after colorectal cancer modeling.

[0021] Figure 8 WT mice after colorectal cancer modeling and Sypl1 - / - Survival status of mice.

[0022] Figure 9 WT mice after colorectal cancer modeling and Sypl1 - / - Changes in the levels of related cytokines in mouse serum.

[0023] Figure 10 WT mice and Sypl1 - / - tropism of mouse BMDMs induced into M1 and M2 macrophages

[0024] Figure 11 WT mice after colorectal cancer modeling and Sypl1 - / - The ratio of M1 and M2 macrophages in mouse intestinal tumor tissue. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1

[0027] A method for constructing a Sypl1 gene knockout mouse model of colorectal cancer, comprising the following steps:

[0028] (1) Sypl1 Gene knockout mouse construction

[0029] Gene knockout strategy: Utilizing CRISPR / Cas-mediated genome engineering technology, gene knockout was performed in C57BL / 6N mice. Sypl1 Gene. Sypl1The gene (NCBI reference sequence: NM\U 013635; Ensembl: ENSMUSG000000020570) is located on mouse chromosome 12 and consists of 6 exons. Exon 3 was selected as the target site for knockout. Figure 1 The gRNA target sequence is as follows:

[0030] gRNA-A1: 5'-ATATGACTCTCGCAATATTT AGG -3' (SEQ ID No.1)

[0031] gRNA-A2: 5'-CTGGGATAAAGCCATCACGC TGG -3' (SEQ ID No. 2)

[0032] Microinjection of fertilized eggs: Three 4-6 week old C57BL / 6N female mice were used. 5 IU of pregnant mare serum gonadotropin (PMSG) was injected intraperitoneally, followed by 5 IU of human chorionic gonadotropin (hCG) 48 hours later. Ovulation was induced 12 hours later. The female mice were then mated with mature male mice. On the fourth day after mating, blastocysts were collected from the donor female mice's uterus. Under a microscope, Cas9 protein (purchased from New England Biolabs, catalog number: M0646) and gRNA were co-injected into the mouse fertilized eggs to construct specific mouse embryonic cells.

[0033] F0 generation mice were obtained by culturing the fertilized eggs in vitro for 1-2 hours, then transferring the surviving fertilized eggs into the oviducts of pseudopregnant mice. Mice born approximately 20 days later were designated as F0 generation mice, and their birth was verified by sequencing. Sypl1 Gene knockout successful ( Figure 2 ).

[0034] Genotyping: Genotyping of F0 generation mice was performed using PCR technology. The primer sequences used are as follows:

[0035] PCR primer 1:

[0036] F1: 5'-TGTGTTGCACTGAGAAATGACC-3' (SEQ ID No.3)

[0037] R1: 5'-TGTTTTGAGTGTTGGCAGCAGATG-3' (SEQ ID No.4)

[0038] PCR primer 2:

[0039] F1: 5'-TGTGTTGCACTGAGAAATGACC-3' (SEQ ID No.5)

[0040] R2: 5'-TTCAGAGAAGACTTGCTGGGC-3' (SEQ ID No.6)

[0041] PCR reaction system: ddH2O 9.0 μL, Primer F 1.0 μL, Primer R 1.0 μL, Premix Taq 12.5 μL, DNA 1.5 μL.

[0042] PCR reaction conditions: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 60℃ annealing for 35 s, 72℃ extension for 35 s, 35 cycles, 72℃ final extension for 5 min.

[0043] The mouse genotype was determined based on the electrophoresis results of the PCR products: Homozygous mice showed one amplified band (427 bp, SEQ ID No. 7) with primer 1, and no amplified band with primer 2; heterozygous mice showed one amplified band (427 bp, SEQ ID No. 7) with primer 1, and one amplified band (646 bp, SEQ ID No. 8) with primer 2; wild-type mice showed no amplified band with primer 1, and one amplified band (646 bp, SEQ ID No. 8) with primer 2. This allows for genotyping of pups. Figure 3 ).

[0044] (2) Constructing a chemically induced mouse model of colorectal cancer

[0045] Using the most commonly used chemical induction modeling method (AOM+DSS), wild-type mice (WT) and gene knockout mice (… Sypl1 - / - A colorectal cancer model was constructed in ).

[0046] Colorectal cancer model group: WT mice and Sypl1 - / - Mice were given a single intraperitoneal injection of AOM (10 mg / kg) and allowed to drink normal water for one week. Then, they were given 2% DSS for one week, followed by two weeks of normal water. This alternating DSS and normal water intake was repeated for three cycles. Tumor formation was monitored using small animal colonoscopy after the three cycles.

[0047] Control group: WT mice and Sypl1 - / - A single intraperitoneal injection of an equal volume of PBS followed by normal drinking water for 10 weeks.

[0048] (3) Evaluation Sypl1 - / - Tumor formation rate in mice

[0049] Using the most commonly used chemically induced modeling method (AOM+DSS), in WT mice and Sypl1 - / - A colorectal cancer model was constructed in mice. For specific modeling methods, please refer to [link to documentation]. Figure 4 A. Throughout the modeling period, mouse body weight was measured periodically. Results showed that, compared to WT mice, Sypl1 - / - The mice lost a significant amount of weight. Figure 4 B). After the model was established, the mice were dissected to measure the length of the colon, and the results showed... Sypl1 - / - The mouse colon is shorter ( Figure 5 ),hint Sypl1 - / - The intestinal damage in mice was more severe. Both small animal colonoscopy and autopsy results showed that, compared to WT mice, Sypl1 - / - After mouse modeling, the number of colorectal tumor lesions increased significantly and the tumor volume was larger. Figure 6 HE staining results also showed Sypl1 - / - The colorectal tumor lesions in mice are larger ( Figure 7 ), indicating knockout Sypl1 The gene significantly increased the tumorigenesis rate of colorectal tumors in mice. Survival analysis showed that compared with WT mice, Sypl1 - / - Mice had shorter lifespans and lower survival rates. Figure 8 The above experimental results show that knocking out mice in vivo... Sypl1 This can promote CRC progress and provide a hint. Sypl1 - / - Mice are an efficient animal model for colorectal cancer.

[0050] (4) Exploration Sypl1 - / - Mechanisms related to increased tumor formation in mice

[0051] To further explore Sypl1 - / - The mechanism by which mice are more prone to tumor formation was investigated by ELISA in WT mice after modeling. Sypl1 - / - The levels of relevant cytokines in mouse serum were compared with those in WT mice. Sypl1 - / - The level of the anti-inflammatory cytokine IL-10 was increased in mouse serum, while the levels of pro-inflammatory cytokines IL-12p70, IL-1β, IL-6, and TNF-α were decreased. Figure 9 Macrophages can exert immunomodulatory effects by secreting a variety of cytokines. Since the aforementioned altered cytokines are the main cytokines secreted during macrophage activation, we further investigated... Sypl1 The effect of gene knockout on macrophage activation. Influenced by the tumor microenvironment, macrophages can differentiate into different types, mainly M1 and M2. M1 macrophages primarily secrete pro-inflammatory cytokines and are generally considered tumor-killing macrophages, mainly playing an anti-tumor and immunomodulatory role; while M2 macrophages primarily secrete anti-inflammatory cytokines, exhibiting immunosuppression and promoting tumor development. Therefore, an increased number of M2 macrophages indicates a poor prognosis. These two types of macrophages have different markers. The commonly used marker for M1 macrophages is CD86, and for M2 macrophages, it is CD206. Therefore, we first used flow cytometry to detect these markers and analyzed the results in WT mice and... Sypl1 - / - BMDMs in mice induced tropism for M1 and M2 macrophages. Results showed that, compared to WT mice, Sypl1 - / - BMDMs in mice are more easily induced into M2 macrophages. Figure 10 Further analysis of the proportions of M1 and M2 macrophages in the intestinal tumor tissue of CRC model mice revealed that... Sypl1 - / - The proportion of M2 / M1 cells in mouse colon tumor tissue was significantly higher than that in WT mice. Figure 11 The above results indicate that knockout Sypl1 Genes can induce macrophages to become M2 polarized, thereby promoting the progression of colorectal cancer.

[0052] In summary, Sypl1 Gene knockout mice can shorten the modeling period of chemically induced colorectal cancer in mice and increase the tumor formation rate of colorectal tumors, making them a highly efficient mouse model for colorectal cancer. Sypl1 - / - The mechanism by which mice are more prone to tumor formation is knockout. Sypl1 Genes can induce macrophages to tend toward M2 polarization.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate and not limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention without departing from the spirit and scope of the present invention. Any modifications or partial substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for constructing a Sypl1 gene knockout mouse model of colorectal cancer, characterized by the following steps: include: (1) Synthesize a gRNA that is a specific target site of the Sypl1 gene; the gRNA includes an anti-strand sequence and a forward sequence; the nucleotide sequence of the anti-strand sequence is shown in SEQ ID No. 1, and the nucleotide sequence of the forward sequence is shown in SEQ ID No. 2; (2) Microinjection: The Cas9 protein, the reverse strand sequence of gRNA and the positive strand sequence of gRNA are mixed to obtain an injection complex, which is microinjected into mouse zygotes. The zygotes are cultured in vitro for 1-2 hours, and the surviving zygotes are transplanted into the oviduct of pseudopregnant mice. Mice born 20 days later are F0 generation mice, which are the Sypl1 gene knockout mouse animal model. (3) A colorectal cancer model was constructed in gene knockout mice using chemical induction modeling method; The inducing agents used in the chemically induced modeling method are azomethane and sodium dextran sulfate.

2. The method according to claim 1, characterized in that, The PCR identification primers for F0 generation mice include PCR primer 1 and PCR primer 2. The nucleotide sequence of the upstream primer of PCR primer 1 is shown in SEQ ID No. 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID No.

4. The nucleotide sequence of the upstream primer of PCR primer 2 is shown in SEQ ID No. 5, and the nucleotide sequence of the downstream primer of primer 2 is shown in SEQ ID No.

6.

3. The application of the Sypl1 gene knockout mouse animal model obtained by the method of any one of claims 1-2 in the study of colorectal cancer, wherein the application does not involve the diagnosis and treatment of the disease.

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