Spontaneous pancreatic cancer mouse model and construction method and application thereof

Abstract

The invention belongs to the technical field of animal model construction, and particularly relates to a spontaneous pancreatic cancer mouse model and a construction method and application thereof. According to the construction method of the spontaneous pancreatic cancer mouse model, the two characteristics of MYC gene overexpression and PTEN gene knockout are applied to the same mouse individual at the same time, the mouse model capable of spontaneously and rapidly forming pancreatic cancer is constructed, the lifetime is short, and the development of related researches of pancreatic cancer is accelerated.

Description

Technical Field

[0001] The invention belongs to the technical field of animal model construction, and specifically relates to a spontaneous pancreatic cancer mouse model and a construction method and application thereof. Background Art

[0002] Pancreatic cancer is one of the most malignant tumors, with an increasing incidence and poor prognosis. Most patients have already lost the opportunity for surgery when diagnosed. At the same time, pancreatic cancer has a poor response to chemotherapy and immunotherapy due to its special tumor microenvironment. Therefore, exploring the formation mechanism of pancreatic cancer microenvironment and finding new therapeutic targets have become urgent issues to be solved in the diagnosis and treatment of pancreatic cancer.

[0003] In recent years, many studies have shown that the proto-oncogene MYC plays a vital role in the development and maintenance of pancreatic cancer. The oncoprotein encoded by MYC can not only regulate the proliferation, differentiation, apoptosis and metabolism of tumor cells themselves, but also form an inhibitory immune microenvironment to cause immune escape, and is usually associated with poor prognosis. MYC is overexpressed or mutated in 70% of tumor patients and is one of the most common highly amplified oncogenes. Different studies have reported that 12% to 15% of pancreatic cancer patients have MYC copy number amplification. PTEN has become one of the important tumor suppressor genes due to its inhibitory effect on the PI3K / AKT pathway. The inactivation of PTEN mainly occurs through allele deletion, gene mutation and methylation modification. In recent years, studies have shown that PTEN inactivation can promote the occurrence and development of various cancers, not only regulating cancer cells themselves, but also affecting various cells in the tumor microenvironment. However, the survival period of mice in the MYC overexpression mouse model and the PTEN gene knockout mouse model is still relatively long, and the cycle from modeling to experiment is long, and the time cost of scientific research is relatively high. Therefore, it is urgent to find a method to construct pancreatic cancer mice with short survival period to accelerate the research progress of scientific researchers. Summary of the invention

[0004] The purpose of the present invention is to provide a spontaneous pancreatic cancer mouse model and its construction method and application, by combining pancreatic specific MYC gene overexpression with PTEN gene knockout mice (LSL-PTEN fl / fl ; LSL-MYC; Pdx1-Cre), shortening the survival time of pancreatic cancer model mice and greatly saving the time and cost of scientific research.

[0005] The present invention provides a method for constructing a spontaneous pancreatic cancer mouse model, comprising: hybridizing a pancreas-specific MYC overexpressing mouse with a pancreas-specific PTEN knockout mouse, and the F1 generation is the spontaneous pancreatic cancer mouse model.

[0006] Preferably, the method for constructing the pancreas-specific PTEN knockout mouse comprises: fl / +Heterozygous positive mice were crossed with pancreas-specifically targeted Pdx1-Cre mice, and the F1 generation was pancreas-specific PTEN knockout mice.

[0007] Preferably, the PTEN fl / + The method for constructing heterozygous positive mice includes: conditional PTEN gene knockout transgenic mice LSL-PTEN fl / + Crossing with C57BL / 6 mice resulted in the PTEN fl / + Heterozygous positive mice.

[0008] Preferably, the conditional PTEN knockout transgenic mouse LSL-PTEN fl / + The construction method includes: C57BL / 6J-Pten fl / + Conditional knockout mice were constructed by knocking out exon 2 on chromosome 19 to obtain conditional PTEN knockout transgenic mice LSL-PTEN fl / + .

[0009] Preferably, the method for constructing a transgenic mouse overexpressing MYC comprises: site-specifically knocking in a CAG-loxp-PGK-Neo-pA-loxp-Myc-WPRE-pA expression frame at the Hipp11 site of chromosome 11 of C57BL / 6J mice to obtain a transgenic mouse overexpressing MYC.

[0010] Preferably, the median survival time of the spontaneous pancreatic cancer mouse model is less than 60 days.

[0011] The present invention also provides the use of the spontaneous pancreatic cancer mouse model constructed by the above construction method in pancreatic cancer drug screening, drug development and / or pancreatic cancer pathogenic mechanism research.

[0012] Beneficial effects: The method for constructing a spontaneous pancreatic cancer mouse model provided by the present invention, based on MYC amplification and PTEN deletion, has been shown in experiments to successfully construct a pancreatic acinar carcinoma combined with ductal carcinoma model in a relatively short period of time, significantly shortening the survival period of the model mice, which can greatly accelerate the research progress of scientific researchers and save time and cost. The spontaneous pancreatic cancer mouse model provided by the present invention has high repeatability and good controllability, and is an important bridge to promote the progress of pancreatic cancer research and its ultimate clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 C57BL / 6J-PTEN fl / +Schematic diagram of the gene knockout region of the conditional knockout mouse; Wild type allele: wild type allele; gRNA region: guide RNA (gRNA) cutting region; targeting vector: target vector; targeted allele: targeted allele; constitutive KO allele (after Cre recombination): knockout (KO, knockout) recombination allele; legends: legends; exon of mouse Pten: mouse PTEN gene exon region; Homology arm: homology arm; cKO region: conditional knockout region; loxP site: loxP insertion site;

[0014] Figure 2 In vitro transcription method for overexpressing MYC mice; Hipp11 genomic loci: hipp11 locus; Wildtype allele: wild-type allele; Targeted allele: targeted allele; Eif4enif1exon19-3'UTR: 3' untranslated region of exon 19 of Eif4enif1 gene; Drg1exon9-3'UTR: 3' untranslated region of exon 9 of Drg1 gene;

[0015] Figure 3 Schematic diagram of in vitro transcription vector; H11 KI vector (myc). Seq: H11 knock-in vector sequence; Kb: kilobase; Amp: ampicillin resistance region; 5'arm: 5' long terminal repeat sequence; 3'arm: 3' long terminal repeat sequence; MC1-hsvTK-polyA: polyoma virus enhancer fused to herpes simplex virus thymidine kinase promoter fused to polyadenylic acid tail; BGHpA: bovine growth hormone polyadenylic acid tail; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element; Myc ORF: MYC gene open coding region; PGK / EM7 / CAG promotor: PGK / EM7 / CAG promoter region; CAintron: CA intron region; Neo: neomycin resistance region; SV40pA: simian vacuolating virus polyadenylic acid tail;

[0016] Figures 4 to 6 This is a schematic diagram of the results of tail identification of F2 generation mice in Example 1;

[0017] Figures 7-10 This is a schematic diagram of the results of tail identification of F2 generation mice in Example 2;

[0018] Figures 11 to 13This is a schematic diagram of the results of tail identification of F1 generation mice in Example 3; Figure 4 , Figure 5 , Fig.11 :Only the lower band is a wild-type mouse, and the upper and lower bands are PTEN heterozygous positive mice; Figure 7 , Figure 8 , Fig.12 :Only the lower band is a wild-type mouse, and the upper and lower bands are MYC overexpression heterozygous positive mice; Figure 6 , Fig. 9 , Fig.10 Those with only the lower band are wild-type mice, and those with the upper and lower bands are Pdx1-Cre single gene heterozygous mice;

[0019] Fig.14 Schematic diagram of spontaneously generated pancreatic cancer tissue in the spontaneous pancreatic cancer mouse model;

[0020] Fig.15 Schematic diagram of HE staining of tumor tissue;

[0021] Fig.16 This is a schematic diagram of the survival time of mice with spontaneous pancreatic cancer;

[0022] Fig.17 This is a schematic diagram comparing the survival time of mice with spontaneous pancreatic cancer and other model mice;

[0023] Fig.18 The median survival time of mice with spontaneous pancreatic cancer is compared with that of other reported mouse models of spontaneous pancreatic cancer. DETAILED DESCRIPTION

[0024] The present invention provides a method for constructing a spontaneous pancreatic cancer mouse model, comprising: hybridizing a pancreas-specific MYC overexpressing mouse with a pancreas-specific PTEN knockout mouse, and the F1 generation is the spontaneous pancreatic cancer mouse model.

[0025] The method for constructing the pancreas-specific PTEN knockout mouse of the present invention preferably comprises: fl / + Heterozygous positive mice were crossed with pancreas-specific Pdx1-Cre tool mice, and the F1 generation was pancreas-specific PTEN knockout mice; the PTEN fl / + The method for constructing heterozygous positive mice preferably comprises: conditionally knocking out the PTEN gene in transgenic mice LSL-PTEN fl / + Crossing with C57BL / 6 mice resulted in the PTEN fl / + Heterozygous positive mice; the conditional PTEN knockout transgenic mice LSL-PTEN fl / + The construction method preferably comprises: C57BL / 6J-Pten fl / +Conditional knockout mice were constructed by knocking out exon 2 on chromosome 19 to obtain conditional PTEN knockout transgenic mice LSL-PTEN fl / + .

[0026] The present invention is for C57BL / 6J-Pten fl / + The sources of conditional knockout mice, pancreas-specific Pdx1-Cre tool mice and C57BL / 6 mice are not particularly limited and can be purchased conventionally. In the embodiments of the present invention, C57BL / 6J-Pten with the item number S-CKO-04509 purchased from Saiye (Suzhou) Biotechnology Co., Ltd. fl / + Conditional knockout mice, pancreas-specific targeted Pdx1-Cre tool mice with strain code 014647 purchased from The Jackson Laboratory China Branch, and C57BL / 6 strain mice with product number NM-CKO-210184 purchased from Shanghai Model Organisms Technology Co., Ltd. are taken as examples, but they cannot be identified as the entire protection scope of the present invention.

[0027] Before constructing the spontaneous pancreatic cancer mouse model described in the present invention, it is preferred to first hybridize the transgenic mice overexpressing MYC with the Pdx1-Cre tool mice with pancreas-specific targeting to obtain the F1 generation mice, which are the pancreas-specific MYC-overexpressing mice described in the present invention; the method for constructing the transgenic mice overexpressing MYC preferably includes site-directed knock-in of the CAG-loxp-PGK-Neo-pA-loxp-Myc-WPRE-pA expression frame at the Hipp11 site of chromosome 11 of C57BL / 6J mice to obtain transgenic mice overexpressing MYC.

[0028] The present invention does not specifically limit the source of the pancreas-specifically targeted Pdx1-Cre tool mouse, which can be purchased conventionally. In the embodiments of the present invention, the pancreas-specifically targeted Pdx1-Cre tool mouse purchased from Shanghai South Model Organisms Technology Co., Ltd. is taken as an example ( Mice and Services, the strain code is 014647), but it cannot be identified as the entire protection scope of the present invention.

[0029] The present invention performs gene identification on the spontaneous pancreatic cancer mouse model obtained, and the identification method is preferably to use PCR to identify PTEN fl / + The tail DNA of the F1 generation obtained by hybridizing heterozygous positive mice with pancreas-specifically targeted Pdx1-Cre positive tool mice was identified.

[0030] The median survival time of the spontaneous pancreatic cancer mouse model of the present invention is less than 60 days. The spontaneous pancreatic cancer of the present invention preferably includes pancreatic acinar carcinoma combined with ductal carcinoma.

[0031] The present invention also provides the use of the spontaneous pancreatic cancer mouse model constructed by the above construction method in pancreatic cancer drug screening, drug development and / or pancreatic cancer pathogenic mechanism research.

[0032] To further illustrate the present invention, a spontaneous pancreatic cancer mouse model provided by the present invention and its construction method and application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] 1. Construction of PTEN knockout mice:

[0035] C57BL / 6J-Pten fl / + Conditional knockout mice (LSL-PTEN fl / + ) was purchased from Saiye (Suzhou) Biotechnology Co., Ltd., with the catalog number S-CKO-04509. The construction process provided by the company includes:

[0036] By consulting the mouse genome information, we found that the PTEN gene is located on mouse chromosome 19, which includes 9 exon regions. We selected exon 2 as the knockout target region (e.g. Figure 1 ), the PTEN protein without this exon region cannot play its biological function;

[0037] RP23-29O22 in the BAC clone library was used as a template to clone the arms of the homologous chromosomes and the knockout region using PCR, and gRNA was obtained and a homologous recombination vector was constructed by in vitro transcription; the gRNA sequence of PTEN was: 5'-AGCAAGGGAAACCTCGCCGAGGG-3' (SEQ ID NO.1); 200 early embryos were obtained by in vitro fertilization of wild-type C57BL / 6J male and female mice; gRNA and homologous recombination vectors were introduced into embryos by microinjection, and the embryos were transplanted into surrogate wild-type female mice. F0 mice were born 3 weeks later, and the genotypes of the grown mice were identified by PCR and sequencing.

[0038] The PCR amplification system is: 1.5 μL of DNA template, 1.0 μL of forward primer, 1.0 μL of reverse primer, 12.5 μL of Taq enzyme and reactant premix, and ddH20 is added to 25 μL; the nucleotide sequence of the forward primer is shown in SEQ ID NO.2, specifically: 5'-CAAGCACTCTGCGAACTGAG-3'; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3, specifically: 5'-AAGTTTTTGAAGGCAAGATGC-3'.

[0039] The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 62°C for 35 s, and extension at 72°C for 35 s, for 33 cycles; maintaining at 72°C for 5 min, and then storing at 4°C for detection.

[0040] The expected results are: the homozygous PTEN knockout shows only one 328 bp band; the heterozygous shows two bands, 328 bp and 156 bp respectively; the wild type shows a 156 bp band.

[0041] The heterozygous mice with the same expected results obtained by the above PCR test were PTEN gene knockout mice (LSL-PTEN fl / + ).

[0042] 2. MYC overexpressing mice (LSL-MYC) were purchased from Shanghai Model Organisms Technology Co., Ltd. with the catalog number NM-CKO-210184. The construction process provided by the company includes:

[0043] By in vitro transcription, the mRNA and gRNA of the Cas9 gene (5'-ATGATGGCATCTAATGAGCTTGG-3', SEQ ID NO. 4) were obtained; by In-Fusion cloning, a homologous recombination vector (donor vector) was constructed, which contained a 5 kb 5' homology arm, CAG-loxp-PGK-Neo-pA-loxp-Myc-WPRE-pA and a 4 kb 3' homology arm, such as Figures 2-3 shown.

[0044] Cas9 mRNA, gRNA and the constructed homologous recombination vector were microinjected into fertilized eggs of C57BL / 6J mice to obtain F0 generation mice. The genotype of F0 generation mice was identified by PCR and sequencing.

[0045] The PCR amplification system was as follows: 2 μL DNA template, 0.8 μL MYC forward primer, 0.8 μL MYC reverse primer, 0.4 μL endogenous control forward primer, 0.4 μL endogenous control reverse primer, 2.4 μL dNTPs, 5.2 μL Taq enzyme and reaction premix, supplemented to 20 μL with ddH20;

[0046] The nucleotide sequence of the MYC forward primer is shown in SEQ ID NO.5, specifically: 5'-TGTCCATTCAAGCAGACGAG-3';

[0047] The nucleotide sequence of the MYC reverse primer is shown in SEQ ID NO.6, specifically: 5'-CCTTGCTCTAGGCAATCCTG-3';

[0048] The nucleotide sequence of the endogenous control forward primer is shown in SEQ ID NO.7, specifically: 5'-CATGCCAATGGTTCACTCTAAGGT-3';

[0049] The nucleotide sequence of the endogenous control reverse primer is shown in SEQ ID NO.8, specifically: 5'-TCTCTATGTCCCAAAGTGCAGACAC-3';

[0050] The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 62°C for 30 s, and extension at 65°C for 82 s, for 35 cycles; maintaining at 72°C for 5 min, and then storing at 4°C for detection.

[0051] The expected result is:

[0052] The wild-type PCR product had no band; the MYC overexpression heterozygote had a 1638 bp band, and the endogenous control had a 335 bp band.

[0053] The heterozygous mice obtained according to the above PCR test with the same expected results are MYC overexpressing mice (LSL-MYC).

[0054] 3. Take a conditional PTEN gene knockout male mouse (LSL-PTEN fl / + ) and mated with 4 C57BL / 6 female mice for 3 weeks to obtain 24 pups. The genotypes of 17 pups were found to have PTEN fl / + The positive rate of heterozygous positive mice was 70.83%, and the theoretical positive rate was 50.00%.

[0055] Then, take one of the PTEN prepared above fl / +Heterozygous positive male mice and 4 pancreas-specific Pdx1-Cre positive tool mice (purchased from The Jackson Laboratory China Branch, The mice were then mated in cages with each other for 3 weeks to obtain 27 F2 mice. The tails of the mice were identified to obtain the genotype results.

[0056] The method for rat tail identification is as follows:

[0057] F2 mice were numbered and their tails were cut 14 days after birth. The required length of the tails was 0.5 cm and they could be stored in a -20°C refrigerator for future use.

[0058] Reagents:

[0059] Rat tail lysis solution: 1M Tris HCl (pH=8.0) 10 ml, 0.5M EDTA 4 ml, 1M NaCl 400 ml, 10% SDS 100 ml, ultrapure water 486 ml.

[0060] Proteinase K solution (PK): 20 mg / ml, stored at -20°C;

[0061] TE buffer: 1M Tris HCL (pH=8.0) 5ml, 0.5M EDTA 1ml, ultrapure water 494ml.

[0062] Experimental process:

[0063] 300 μl of lysis buffer and 5 μl of PK were added to each of the 27 mouse tail samples, and digested overnight at 55°C. After overnight digestion, 600 μl of -20°C precooled anhydrous ethanol was added to each of the digested samples, and the samples were inverted until flocculent precipitates could be clearly seen, and then the inversion was stopped and centrifuged at 12,000 rpm for 5 minutes.

[0064] After centrifugation, pour out the upper liquid and place the centrifuge tube vertically upside down on a piece of flat paper. After maintaining for 10 minutes, change the upright tube to another position, press down lightly, and wait until it is completely dry. At this time, the precipitate becomes transparent and the extracted DNA is of good quality.

[0065] Add 200 μl of TE buffer to the centrifuge tube, shake up and down to fully dissolve the DNA to obtain a DNA template; take 3 μl of the DNA template and perform PCR in a 25 μl reaction system.

[0066] The PCR amplification system is: 3 μL of DNA template, 0.5 μL of forward primer F, 0.5 μL of reverse primer R, 12.5 μL of Taq enzyme and reaction premix, and ddH20 is added to 25 μL; the primer information used is the same as steps 1 and 2, SEQ ID No. 2 and SEQ ID No. 3 are used for PTEN gene identification primers, and SEQ ID No. 5-8 are used for MYC identification primers; the Pdx1-Cre primer sequences are as follows: F: 5'-CCTGGACTACATCTTGAGTTGC-3' (SEQ ID No. 9); R: 5'-AGGCAAATTTTGGTGTACGG-3' (SEQ ID No. 10); Pdx1-Cre expected results: the wild type shows only one 200 bp band; the heterozygote shows two bands, 650 bp and 200 bp, respectively.

[0067] The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 95°C for 30 s, annealing at 65°C for 30 s, and extension at 72°C for 35 s, for 10 cycles; denaturation at 95°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for 25 cycles; hold at 72°C for 5 min, and then store at 4°C for testing;

[0068] Electrophoresis: Prepare 1-3% agarose gel according to the target band, dissolve 1g-3g agarose in 100ml TAE buffer, heat in a microwave until the agarose melts, add 10μl nucleic acid dye (EB substitute), pour into the electrophoresis template, insert the comb, and use it after condensation; add an appropriate amount of DNA to the agarose gel, add 10μl to the small hole and 20μl to the large hole, and select a suitable DNA marker according to the size of the target band and add it to the gel. After running the gel at 120V voltage, use the gel imaging system to observe and take photos to obtain the genotype identification result report. The gene identification results of 27 F2 mice are as follows: Figures 4 to 6 As shown, Figure 4 Maker and Figure 5 The consistency in .

[0069] Depend on Figures 4-5 The results showed that PTEN fl / + There were 13 single gene heterozygous mice, with a positive rate of 48.15% and a theoretical positive rate of 50.00%. Figure 6 The results showed that there were 15 Pdx1-Cre single gene heterozygous mice, with a positive rate of 55.56% and a theoretical positive rate of 50.00%. Figures 4 to 6 The results of gene identification of 27 F2 mice showed that 8 of them showed PTEN fl / + ; Pdx1-Cre double heterozygous positive.

[0070] Example 2

[0071] One conditional MYC gene overexpressing male mouse (LSL-MYC) obtained in step 2 of Example 1 was selected and mated with four C57BL / 6 female mice for 3 weeks to obtain 31 pups. Their genotypes were identified by the mouse tail identification method in Example 1, and 20 LSL-MYC heterozygous positive mice were found, with a positive rate of 64.52% and a theoretical positive rate of 50.00%.

[0072] One of the LSL-MYC heterozygous positive male mice was mated with four pancreas-specific Pdx1-Cre positive mice for 3 weeks to obtain 25 F2 mice. The tails of the 25 F2 mice were identified according to the method in Example 1. The gene identification results of the 25 F2 mice are as follows: Figures 7-10 As shown, Figure 8 Maker and Figure 7 The consistency in .

[0073] Depend on Figures 7-8 The results showed that there were 15 LSL-MYC single gene heterozygous mice, with a positive rate of 60.00% and a theoretical positive rate of 50.00%. Figures 9-10 The results showed that there were 11 Pdx1-Cre single gene heterozygous mice, with a positive rate of 44.00% and a theoretical positive rate of 50.00%. Figures 7-10 According to the results, among the 25 F2 mice, 7 were LSL-MYC and Pdx1-Cre double heterozygous positive.

[0074] Example 3

[0075] Take one PTEN obtained in Example 1 fl / + The LSL-MYC / Pdx1-Cre double gene heterozygous male mice were mated with the three LSL-MYC / Pdx1-Cre double gene heterozygous female mice obtained in Example 2 for 3 weeks to obtain 24 F1 pups. The tails of the mice were identified according to the method in Example 1. The results are as follows: Figures 11 to 13 shown.

[0076] Depend on Fig.11 The results showed that PTEN fl / + There were 17 single gene heterozygous mice, with a positive rate of 70.83% and a theoretical positive rate of 50.00%. Fig.12 The results showed that there were 19 LSL-MYC single gene heterozygous mice, with a positive rate of 79.17% and a theoretical positive rate of 50.00%. Fig.13 The results showed that there were 11 Pdx1-Cre single gene heterozygous mice, with a positive rate of 45.83% and a theoretical positive rate of 50.00%. Figures 11 to 13 In 24 F1 mice, PTEN fl / + / LSL-MYC; Pdx1-Cre triple heterozygous genotype 10 mice, the positive rate was 41.67%, and the theoretical positive rate was 12.50%. fl / + The / LSL-MYC / Pdx1-Cre triple heterozygous genotype mouse is the spontaneous pancreatic cancer mouse model described in the present invention, also known as PMC mouse.

[0077] Example 4

[0078] 18 PMC mice were constructed by the methods of Examples 1 to 3. After the genotype was confirmed by tail identification, the mice were routinely raised in an SPF animal room. The mice were observed once a week within 4 weeks of age and every two days after 4 weeks of age. When the mice were emaciated, listless, or had decreased activity, aseptic laparotomy was performed to explore the pancreatic tissue to determine whether there was a spontaneous pancreatic tumor. If a tumor was present, the mouse was killed, and the tumor was removed and gross photographs were taken (such as Fig.14 ) and preserved in formalin for subsequent paraffin embedding and sectioning for HE staining or immunohistochemistry experiments (such as Fig.15 As shown, the left side is the electron micrograph of pancreatic acinar carcinoma, and the right side is the electron micrograph of pancreatic ductal carcinoma). At the same time, the survival time of mice on the day of sacrifice was recorded to determine the median survival time of PMC mice (as shown in Fig.16 shown).

[0079] Depend on Fig.14 It can be seen that the model mice constructed by the present invention can spontaneously and rapidly develop pancreatic tumors; Fig.15 It can be seen that the pancreatic tumors formed by the model mice constructed by the present invention have two pathological types, acinar carcinoma and ductal carcinoma, which can represent the rare pancreatic cancer patients with both pathological types encountered in clinical practice. Such pancreatic cancer patients usually have a rapid disease progression and a poor prognosis, which is consistent with the pathological characteristics of this mouse model. Therefore, this model has a certain clinical representativeness and is suitable for laboratory research. In addition, the construction of this model is the first in the world and has not been reported before.

[0080] Comparative Example 1

[0081] The four PTEN constructs in Example 1 fl / + / Pdx1-Cre double heterozygous mice, 15 LSL-Myc / Pdx1-Cre double heterozygous mice constructed in Example 2, 18 PMC mice constructed in Example 4, and 1 Pdx1-Cre mouse purchased were routinely raised in an SPF animal room after their genotypes were accurately identified by the mouse tail identification method in Example 1. The health status of the mice was observed once a week within 4 weeks of age, and every two days after 4 weeks of age. When the mice showed signs of emaciation, lack of energy, decreased activity, etc., aseptic laparotomy was performed to explore the pancreatic tissue to determine whether there was a spontaneous pancreatic tumor. If a pancreatic tumor was present, the mouse was euthanized and the survival time of the mouse was recorded. Fig.17 shown.

[0082] At the same time, the survival curve of PMC mice was compared with the survival curve of other mice with spontaneous pancreatic cancer (all of which were constructed and cultured by the applicant according to the literature to obtain the above experiment results). It was found that the median survival time of PMC mice was significantly shorter than that of several other mouse models, such as Fig.18 As shown; the other literatures reported on the survival curves of mice with spontaneous pancreatic cancer are as follows: [Sunil R Hingorani, et al. Trp53R172H and KrasG12D cooperate topromote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell. 2005 May; 7(5): 469-83.], [Beatriz Salvador-Barbero, et al. CDK4 / 6 Inhibitors Impair Recovery from Cytotoxic Chemotherapy in Pancreatic Adenocarcinoma. Cancer Cell. 2020 Mar 16; 37(3): 340-353. e6.] and [Janel L Kopp, et al. Loss of Pten and Activation of Kras Synergistically Induce Formation of Intraductal Papillary Mucinous Neoplasia From Pancreatic Ductal Cells in Mice.Gastroenterology.2018Apr;154(5):1509-1523.e5.].

[0083] Depend on Fig.17 and Fig.18 It can be seen that the model mice constructed by the present invention have a shorter median survival time than other model mice, which saves modeling time, is more conducive to accelerating scientific research progress and saving scientific research time.

[0084] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing a spontaneous pancreatic cancer mouse model, characterized in that: include: Mice that overexpress MYC in the pancreas are hybridized with mice that knock out PTEN in the pancreas, and the F1 generation is the spontaneous pancreatic cancer mouse model.

2. The construction method according to claim 1, characterized in that: The method for constructing a pancreas-specific PTEN knockout mouse comprises: fl / + Heterozygous positive mice were crossed with pancreas-specifically targeted Pdx1-Cre mice, and the F1 generation was pancreas-specific PTEN knockout mice.

3. The construction method according to claim 2, characterized in that: PTEN fl / + The method for constructing heterozygous positive mice includes: conditional PTEN gene knockout transgenic mice LSL-PTEN fl / + Crossing with C57BL / 6 mice resulted in the PTEN fl / + Heterozygous positive mice.

4. The construction method according to claim 3, characterized in that: The conditional PTEN knockout transgenic mouse LSL-PTEN fl / + The construction method includes: C57BL / 6J-Pten fl / + Conditional knockout mice were constructed by knocking out exon 2 on chromosome 19 to obtain conditional PTEN knockout transgenic mice LSL-PTEN fl / + .

5. The construction method according to claim 1, characterized in that: The method for constructing the pancreas-specific MYC-overexpressing mouse comprises: hybridizing the MYC-overexpressing transgenic mouse with the pancreas-specifically targeted Pdx1-Cre tool mouse, and the F1 generation is the pancreas-specific MYC-overexpressing mouse.

6. The construction method according to claim 5, characterized in that: The method for constructing a transgenic mouse overexpressing MYC comprises: site-specifically knocking in a CAG-loxp-PGK-Neo-pA-loxp-Myc-WPRE-pA expression frame at the Hipp11 site of chromosome 11 of a C57BL / 6J mouse to obtain a transgenic mouse overexpressing MYC.

7. The construction method according to any one of claims 1 to 6, characterized in that: The median survival time of the spontaneous pancreatic cancer mouse model is less than 60 days.

8. Use of the spontaneous pancreatic cancer mouse model constructed according to the construction method according to any one of claims 1 to 7 in pancreatic cancer drug screening, drug development and / or pancreatic cancer pathogenesis research.

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