Establishment method and application of mouse gastric cancer model

Abstract

The invention belongs to the technical field of biotechnology, and discloses an establishment method and application of a mouse gastric cancer model, which not only can further reveal the biological mechanism of Tibetan in Qinghai to adapt to hypoxia, but also has important reference significance for developing therapeutic target drugs for preventing and treating digestive tract tumor diseases of plateau crowds. According to data provided by NCBI (National Center of Biotechnology Information), the HIF-1alpha gene is located on a chromosome 14, the human-derived HIF-1alpha and the mouse HIF-1alpha both have an encodable transcript, the corresponding protein transcript numbers are respectively NP001834.1 and NP031609.5, and the homology of the human-derived HIF-1alpha and the mouse HIF-1alpha is 95.3% through comparison. The amino acid at the human 582nd site is proline, and the target mutation site of the mouse HIF-1alpha gene is also located at the 582nd site, so that a corresponding gene editing mouse is designed on the site.

Description

Technical Field

[0001] The invention belongs to the technical field of biotechnology, and in particular relates to a method for establishing a mouse gastric cancer model and an application thereof. Background Art

[0002] The average altitude of the Qinghai-Tibet Plateau is above 4,000 meters. The Tibetan people have lived on the Qinghai-Tibet Plateau for at least 20,000 to 30,000 years, making them the best subjects for studying plateau acclimatization. Hypoxia is a typical climatic feature of the Qinghai-Tibet Plateau and one of the microenvironmental characteristics of tumors. It is not only the initiating factor for tumor deterioration, but also accelerates angiogenesis in tumor tissues. Hypoxia-inducible factor-1α (HIF-1α) is the earliest transcriptional regulator produced when the body encounters hypoxia, and is also the core promoter of new blood vessel formation. Studies have found that the ability of HIF-1α and the hypoxia response element (HRE) complex to activate vascular endothelial growth factor (VEGF) is enhanced under hypoxia, indicating that hypoxia promotes microvascular growth by upregulating the expression of VEGF. At the same time, after binding to p53, HIF-1α inhibits the unlimited growth of microvessels in a hypoxic environment by enhancing the transcription of downstream target genes related to angiogenesis inhibition. In 2001, Clifford first discovered that the rs11549465 (C1772T, P582S) single nucleotide polymorphism (SNP) located in the oxygen-dependent degradation region (ODD) of the HIF-1a gene was positively correlated with tumor susceptibility. Subsequently, a large number of studies on HIF-1αrs11549465 found that this SNP site significantly enhanced the transcriptional activity of HIF-1α and multiple downstream genes under normoxic and hypoxic conditions; and by increasing microvascular density, it broke the balance of angiogenesis and promoted the development of tumors. Many meta-analyses at home and abroad have shown that this SNP site is more commonly found in high-altitude populations in Asia and plays an important role in the formation of digestive system tumors. The mortality rate of gastric cancer ranks first among all cancers in my country (40.62 / 100,000). Qinghai is one of the high-incidence areas of gastric cancer in my country. The incidence of gastric cancer in the Tibetans in Qinghai is significantly higher than that in other provinces and ethnic groups, and they have a higher Helicobacter pylori (HP) infection rate. In 1994, the International Cancer Organization officially announced that HP is a Class I carcinogen for gastric cancer. It is the only prokaryotic organism that has been clearly classified as a carcinogen. The chance of developing gastric cancer after infection increases by 4-9 times, and more than 50% of gastric cancer patients are found to be infected with HP after examination. According to the presence of toxin-related gene A (CagA) protein, HP is divided into type I (CagA+) and type II (CagA-), and type I can cause more severe gastric mucosal damage and a higher incidence of gastric cancer than type II. Through the experiment of H (CagA+) long-term infection-induced gastric cancer model in mice, it was found that the microvascular density of gastric cancer tissue was significantly higher than that of normal tissue: and it was found that Hp (CagA+) stabilized the expression of HIF-1α under normoxic conditions and affected its downstream signaling pathways. Therefore, in-depth research on the role of HTF-1αrs11549465 in gastric cancer in the Tibetan population living in Qinghai and its mechanism of action with H susceptibility can not only further reveal the biological mechanism of Qinghai Tibetans' adaptation to hypoxia, but also have important reference significance for the development of therapeutic target drugs for the prevention and treatment of gastrointestinal tumor diseases in plateau populations.

[0003] Through the above analysis, the problems and defects of the prior art are as follows:

[0004] The role of HTF-1αrs11549465 in gastric cancer in the Tibetan population living in Qinghai and its mechanism of action with Hp susceptibility are unclear. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a method for establishing a mouse gastric cancer model and application thereof.

[0006] The present invention is achieved by: a method for establishing a mouse gastric cancer model comprising:

[0007] Step 1, selection of experimental mice: mice with C57BL / 6 background and their fertilized eggs were selected;

[0008] Step 2: Obtaining genetic information:

[0009] Obtain human and mouse HIF-1α gene sequence, protein sequence and domain information from NCBI and Ensembl websites, perform homology comparisons, and obtain HIF-1α gene transcript information; obtain current HIF-1α mutant mouse model information and prediction information on the phenotype caused by the gene mutation from the MGI website;

[0010] Step 3, construction of gene-edited mice;

[0011] Vector construction:

[0012] According to the design plan, design, construct and in vitro transcribe sgRNA, construct the source recombinant vector, and verify the correctness of the vector sequence by sequencing;

[0013] Embryo microinjection:

[0014] Cas9, sgRNA and Donor vector samples were microinjected into fertilized eggs of mice with C57BL / 6 background; fertilized eggs that survived the injection were transplanted into pseudo-pregnant female mice and waited for them to become pregnant and give birth;

[0015] Identification of F0 mice:

[0016] The F0 generation mice pups born to the recipient mice were tail-clipped and toe-clipped at 5-7 days old, and their genomic DNA was extracted for PCR and sequencing to determine their genotypes;

[0017] F0 mouse breeding and F1 mouse identification; mouse breeding conditions.

[0018] Further, the identification method of the F0 mice is as follows:

[0019] (1) DNA extraction from mouse tissues;

[0020] (2) Take the extracted mouse tissue DNA and send it to the first-generation sequencing to obtain the gene sequence to confirm whether the editing is successful.

[0021] Further, the mouse tissue DNA extraction method:

[0022] 1) Reagents and consumables:

[0023] Lysis buffer: TailDigestionBuffer (500ml): 1M TrisHCl (PH=8.0) 25ml, 0.5M EDTA 100ml, 5M NaCl 10ml, 20% SDS 25ml, MillQ 340ml, containing proteinase K (PK, 10mg / ml); 100% ethanol; 70% ethanol; TEBuffer (500ml): 1M Tris-HCl (PH=8.0) 5ml, 0.5M EDTA (PH=8.0) 1ml, MillQ 494ml, phenol and chloroform 1:1 mixed solution;

[0024] 2) Take the mouse tail and put it into a 1.5 ml EP tube;

[0025] 3) Prepare lysis buffer: add 5ul proteinase K to every 500ul lysis buffer, and dispense 500ul into each EP tube containing the tail;

[0026] 4) Place the EP tube with lysis solution on the EP tube rack, wrap it with plastic wrap and place it on a metal heating block at 55°C for overnight digestion;

[0027] 5) The next day, shake the EP tube vigorously to make the digested tissue uniform;

[0028] 6) Add 500ul of an equal volume of phenol / chloroform mixed solution to each tube;

[0029] 7) Mix vigorously and centrifuge at 12000 rpm for 15 min;

[0030] 8) Use a 1000ul pipette to draw about 300ul of supernatant from the centrifuged EP tube into a new EP tube and mark the number clearly. When drawing the supernatant, be careful not to draw the middle layer or the lower layer;

[0031] 9) Add 100% ethanol twice the volume of the supernatant (400ul) to each tube, mix by inverting, white filamentous DNA should be visible, and centrifuge at 12000rpm for 10min;

[0032] 10) Discard the supernatant, add 800ul70% ethanol, and centrifuge at 12000rpm for 5min;

[0033] 11) Discard the supernatant, carefully absorb the residual ethanol, and dry it until there is no alcohol smell;

[0034] 12) Add 100ul TE solution to dissolve the DNA. The solution can be dissolved at 55℃ or 37℃ and stored at -20℃ for a long time.

[0035] Furthermore, the 1:1 mixed solution of phenol and chloroform was stored at 4°C;

[0036] The length of the mouse tail does not exceed 0.3 cm;

[0037] Drying time is more than 30 minutes.

[0038] Furthermore, the F0 mouse breeding and F1 mouse identification:

[0039] After the positive F0 generation mice reached sexual maturity, they were mated with wild-type C57BL / 6 background mice; the tails of the F1 generation mice born were cut and the toes were numbered on days 5-7, and genomic DNA was extracted for PCR and sequencing identification;

[0040] 1) The mice obtained by targeting with the CRISPR / Cas9 system are affected by the sgRNA cutting efficiency and donor recombination efficiency, and multiple genotypes coexist in the F0 generation;

[0041] 2) After backcrossing positive F0 mice with wild-type background mice, the resulting offspring will have a single genotype;

[0042] 3) F0 generation mice cannot mate with each other;

[0043] After obtaining the F1 mice, their genotypes were identified using the same identification system as that of the F0 mice.

[0044] Further, the mouse breeding conditions are:

[0045] 1) The mice used in this experiment were raised in an SPF animal room.

[0046] 2) The temperature in the breeding room is maintained at 22-28℃, the relative humidity is maintained at 40%-60%, the light and dark alternation time is 12 / 12, and the noise is less than 60dB

[0047] 3) Feed: Sterile complete nutritional granules sterilized by 60Co gamma rays

[0048] 4) Drinking water: Bottled pure water is sterilized under high pressure and the water is changed once a week to ensure continuous drinking water supply

[0049] 5) Bedding: Use autoclaved mixed sawdust, change once a week

[0050] 6) Daily registration and observation: register various environmental data and mouse conditions every day, and closely observe the mice's diet, activities, and overall condition;

[0051] 7) Tail and toe clipping and numbering can be performed on the pups 5-7 days after birth; ear tagging and cage separation can be performed on the pups 19-21 days after birth. All operations are performed in a clean bench in accordance with sterile procedures.

[0052] Another object of the present invention is to provide a system for establishing a mouse gastric cancer model comprising:

[0053] Selection module, used to select mice with C57BL / 6 background and their fertilized eggs;

[0054] Gene information acquisition module, used to obtain human and mouse HIF-1α gene sequence, protein sequence and domain information from NCBI and Ensembl websites, and perform homology comparison to obtain transcript information of HIF-1α gene; obtain current HIF-1α mutant mouse model information and prediction information on the phenotype caused by the gene mutation from the MGI website;

[0055] Building blocks for gene-edited mouse construction.

[0056] Another object of the present invention is to provide a computer device, comprising a memory and a processor, wherein the memory stores a computer program, and when the computer program is executed by the processor, the processor executes the steps of the method for establishing a mouse gastric cancer model.

[0057] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, wherein when the computer program is executed by a processor, the processor executes the steps of the method for establishing a mouse gastric cancer model.

[0058] Another object of the present invention is to provide an information data processing terminal, which is used to implement the system for establishing the mouse gastric cancer model.

[0059] In combination with the above technical solutions and the technical problems solved, please analyze the advantages and positive effects of the technical solutions to be protected by the present invention from the following aspects:

[0060] First, in view of the technical problems existing in the above-mentioned prior art and the difficulty of solving the problems, the technical solutions to be protected by the present invention and the results and data during the research and development process are closely combined to analyze in detail and deeply how the technical solutions of the present invention solve the technical problems, and some creative technical effects brought about after solving the problems. The specific description is as follows:

[0061] The present invention deeply studies the role of HIF-1αrs11549465 in gastric cancer in the Tibetan population living in Qinghai and its mechanism of action with Hp susceptibility, which can not only further reveal the biological mechanism of Qinghai Tibetans' adaptation to hypoxia, but also has important reference significance for the development of therapeutic target drugs for the prevention and treatment of gastrointestinal tumor diseases in plateau populations.

[0062] HIF-1α gene information: According to the data provided by NCBI, the HIF-1α gene is located on chromosome 14. Both human and mouse HIF-1α have an coded transcript, and the corresponding protein transcript numbers are NP_001834.1 and NP_031609.5, respectively. After comparison, the homology between the two is 95.3%. The amino acid at position 582 of human is proline, and the target mutation site of the mouse HIF-1α gene is also located at amino acid position 582, so the corresponding gene-edited mouse is designed at this site.

[0063] Mutation site of HIF-1α mutant mice: According to the data provided by the Ensembl website, HIF-1α has two available transcripts. Among them, the transcript numbered HIF-1α-185 (ENSMUST00000059767.15) has the highest score. This transcript encodes 13 exons, the transcript is 15766bp long, and encodes 1222 amino acids.

[0064] Second, the technical solution of the present invention solves several key problems related to the establishment of mouse gastric cancer models in the prior art in industrial applications, and has achieved significant technical progress, which is specifically reflected in the following aspects:

[0065] 1. Accurately simulate the occurrence mechanism of human gastric cancer

[0066] Most existing mouse gastric cancer models rely on chemical induction or viral infection. Although these methods can induce gastric cancer, they are quite different from the genetic pathogenic mechanisms of human gastric cancer, which limits their application in precision medicine research. The present invention uses CRISPR / Cas9 gene editing technology to accurately knock out or mutate key genes (such as HIF-1α) in mice, and successfully constructs a mouse model that can better simulate the genetic mutation of human gastric cancer. This gene editing model can accurately simulate the genetic mechanism of human gastric cancer, making it have a higher reference value in gastric cancer research, drug screening and targeted therapy.

[0067] 2. Improve the reliability and repeatability of the model

[0068] The traditional method for establishing a mouse gastric cancer model is unstable and inconsistent, resulting in poor repeatability of experimental results and increased research costs. The present invention introduces gene editing technology and adopts standardized mouse fertilized egg injection and subsequent genotype identification procedures to greatly improve the success rate and controllability of the model. Through PCR and sequencing identification of genotypes, the genotypes of F0 and F1 generations of mice are ensured to be consistent, so that the experimental model is more consistent in different experimental batches, and the reliability and repeatability of the research results are improved.

[0069] 3. Accelerate drug screening and treatment research related to gastric cancer

[0070] The applicability of gastric cancer models in the prior art in drug screening and targeted therapy research is relatively limited, especially when exploring drugs targeting specific gene mutations, it is difficult to provide effective data support. The present invention introduces HIF-1α and other gastric cancer-related mutant genes into mice to establish a model that is closer to the actual pathological process, providing an ideal experimental platform for drug screening, targeted therapy, immunotherapy and other research. This model can effectively verify the efficacy and safety of drugs, accelerate the process of drug development, and enhance the value of industrial application.

[0071] 4. Efficient gene editing and rapid model construction

[0072] Compared with the traditional method of building mouse models, which is time-consuming and inefficient, the present invention significantly shortens the construction time of mouse gastric cancer models through the CRISPR / Cas9 system. Gene editing technology can achieve precise modification of target genes in a short period of time, greatly reducing the mouse breeding time required for the experiment and improving the success rate of the model. This method of rapid model construction has an important role in accelerating cancer research and preclinical research.

[0073] 5. Wide applicability of the model

[0074] The technical solution of the present invention can not only be used for gastric cancer research, but also be extended to other cancer research related to gene mutations. By adjusting the design of sgRNA, gene-edited mouse models for different cancer types can be quickly constructed. This flexibility and versatility provides broad application prospects for cancer research and has significant industrialization potential.

[0075] In summary, the technical solution of the present invention successfully solves the problems of model instability and inconsistency with human pathological mechanisms in the prior art through precise gene editing methods, and has made significant technological progress in gastric cancer research, drug screening and cancer treatment, greatly improving the efficiency and accuracy of experimental research. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1It is a flow chart of the method for establishing a mouse gastric cancer model provided in an embodiment of the present invention.

[0077] Figure 2 It is a flow chart of the identification method of F0 mice provided in an embodiment of the present invention.

[0078] Figure 3 It is a structural block diagram of a system for establishing a mouse gastric cancer model provided by an embodiment of the present invention.

[0079] Figure 4 It is a diagram of the mouse genotype PCR identification strategy provided in an embodiment of the present invention.

[0080] Figure 5 This is a 36-week HE staining image provided by an embodiment of the present invention.

[0081] Figure 6 This is a 36-week Giemsa staining image provided by an embodiment of the present invention.

[0082] Figure 7 This is a 36-week CD34 graph provided by an embodiment of the present invention.

[0083] Figure 8 This is a 36-week PCR chart provided in an embodiment of the present invention.

[0084] Fig. 9 It is a 36-week WB chart provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0085] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0086] like Figure 1 As shown, a method for establishing a mouse gastric cancer model provided in an embodiment of the present invention comprises the following steps:

[0087] S101, selection of experimental mice; mice with C57BL / 6 background and their fertilized eggs were selected;

[0088] S102, Acquisition of Gene Information:

[0089] Obtain human and mouse HIF-1α (hypoxia-inducible factor-1α) gene sequence, protein sequence and domain information from NCBI and Ensembl websites, perform homology comparisons, and obtain HIF-1α gene transcript information; obtain current HIF-1α mutant mouse model information and prediction information on the phenotype caused by the gene mutation from the MGI (Mouse Genome Informatics) website;

[0090] S103, gene-edited mouse construct;

[0091] Vector construction:

[0092] According to the design plan, design, construct and in vitro transcribe sgRNA, construct the source recombinant vector, and verify the correctness of the vector sequence by sequencing;

[0093] Embryo microinjection:

[0094] Cas9, sgRNA and Donor vector samples were microinjected into fertilized eggs of mice with C57BL / 6 background; fertilized eggs that survived the injection were transplanted into pseudo-pregnant female mice and waited for them to become pregnant and give birth;

[0095] Identification of F0 mice:

[0096] The F0 generation mice pups born to the recipient mice were tail-clipped and toe-clipped at 5-7 days old, and their genomic DNA was extracted for PCR and sequencing to determine their genotypes;

[0097] F0 mouse breeding and F1 mouse identification; mouse breeding conditions.

[0098] like Figure 2 As shown, the identification method of F0 mice provided in the embodiment of the present invention is as follows:

[0099] S201, DNA extraction from mouse tissues;

[0100] S202, taking the extracted mouse tissue DNA and sending it to first-generation sequencing to obtain the gene sequence to confirm whether the editing is successful.

[0101] The method for extracting DNA from mouse tissue provided by the embodiment of the present invention is as follows:

[0102] 1) Reagents and consumables:

[0103] Lysis buffer: TailDigestionBuffer (500ml): 1M TrisHCl (PH=8.0) 25ml, 0.5M EDTA 100ml, 5M NaCl 10ml, 20% SDS 25ml, MillQ 340ml, containing proteinase K (PK, 10mg / ml); 100% ethanol; 70% ethanol; TEBuffer (500ml): 1M Tris-HCl (PH=8.0) 5ml, 0.5M EDTA (PH=8.0) 1ml, MillQ 494ml, phenol and chloroform 1:1 mixed solution;

[0104] 2) Take the mouse tail and put it into a 1.5 ml EP tube;

[0105] 3) Prepare lysis buffer: add 5ul proteinase K to every 500ul lysis buffer, and dispense 500ul into each EP tube containing the tail;

[0106] 4) Place the EP tube with lysis solution on the EP tube rack, wrap it with plastic wrap and place it on a metal heating block at 55°C for overnight digestion;

[0107] 5) The next day, shake the EP tube vigorously to make the digested tissue uniform;

[0108] 6) Add 500ul of an equal volume of phenol / chloroform mixed solution to each tube;

[0109] 7) Mix vigorously and centrifuge at 12000 rpm for 15 min;

[0110] 8) Use a 1000ul pipette to draw about 300ul of supernatant from the centrifuged EP tube into a new EP tube and mark the number clearly. When drawing the supernatant, be careful not to draw the middle layer or the lower layer;

[0111] 9) Add 100% ethanol twice the volume of the supernatant (400ul) to each tube, mix by inverting, white filamentous DNA should be visible, and centrifuge at 12000rpm for 10min;

[0112] 10) Discard the supernatant, add 800ul70% ethanol, and centrifuge at 12000rpm for 5min;

[0113] 11) Discard the supernatant, carefully absorb the residual ethanol, and dry it until there is no alcohol smell;

[0114] 12) Add 100ul TE solution to dissolve the DNA. The solution can be dissolved at 55℃ or 37℃ and stored at -20℃ for a long time.

[0115] The 1:1 mixed solution of phenol and chloroform provided in the embodiment of the present invention is stored at 4° C.;

[0116] The length of the mouse tail does not exceed 0.3 cm;

[0117] Drying time is more than 30 minutes.

[0118] F0 mouse breeding and F1 mouse identification provided by the embodiments of the present invention:

[0119] After the positive F0 generation mice reached sexual maturity, they were mated with wild-type C57BL / 6 background mice; the tails of the F1 generation mice born were cut and the toes were numbered on days 5-7, and genomic DNA was extracted for PCR and sequencing identification;

[0120] 1) The mice obtained by targeting with the CRISPR / Cas9 system are affected by the sgRNA cutting efficiency and donor recombination efficiency, and multiple genotypes coexist in the F0 generation;

[0121] 2) After backcrossing positive F0 mice with wild-type background mice, the resulting offspring will have a single genotype;

[0122] 3) F0 generation mice cannot mate with each other;

[0123] After obtaining the F1 mice, their genotypes were identified using the same identification system as that of the F0 mice.

[0124] The mouse breeding conditions provided in the embodiment of the present invention are:

[0125] 1) The mice used in this experiment were raised in an SPF animal room.

[0126] 2) The temperature in the breeding room is maintained at 22-28℃, the relative humidity is maintained at 40%-60%, the light and dark alternation time is 12 / 12, and the noise is less than 60dB

[0127] 3) Feed: Sterile complete nutritional granules sterilized by 60Co gamma rays

[0128] 4) Drinking water: Bottled pure water is sterilized under high pressure and the water is changed once a week to ensure continuous drinking water supply

[0129] 5) Bedding: Use autoclaved mixed sawdust, change once a week

[0130] 6) Daily registration and observation: register various environmental data and mouse conditions every day, and closely observe the mice's diet, activities, and overall condition;

[0131] 7) Tail and toe clipping and numbering can be performed on the pups 5-7 days after birth; ear tagging and cage separation can be performed on the pups 19-21 days after birth. All operations are performed in a clean bench in accordance with sterile procedures.

[0132] like Figure 3As shown, a system for establishing a mouse gastric cancer model provided by an embodiment of the present invention includes:

[0133] Selection module, used to select mice with C57BL / 6 background and their fertilized eggs;

[0134] Gene information acquisition module, used to obtain human and mouse HIF-1α gene sequence, protein sequence and domain information from NCBI and Ensembl websites, and perform homology comparison to obtain transcript information of HIF-1α gene; obtain current HIF-1α mutant mouse model information and prediction information on the phenotype caused by the gene mutation from the MGI website;

[0135] Building blocks for gene-edited mouse construction.

[0136] Another object of the present invention is to provide a computer device, comprising a memory and a processor, wherein the memory stores a computer program, and when the computer program is executed by the processor, the processor executes the steps of the method for establishing a mouse gastric cancer model.

[0137] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, wherein when the computer program is executed by a processor, the processor executes the steps of the method for establishing a mouse gastric cancer model.

[0138] Another object of the present invention is to provide an information data processing terminal, which is used to implement the system for establishing the mouse gastric cancer model.

[0139] The present invention is specifically implemented:

[0140] 1.1 Selection of experimental mice

[0141] C57BL / 6 background mice and their fertilized eggs were selected.

[0142] 1.2 Acquisition of genetic information

[0143] The human and mouse HIF-1α gene sequence, protein sequence and domain information were obtained from NCBI and Ensembl websites, and homology comparison was performed to obtain the transcript information of the HIF-1α gene; the current HIF-1α mutant mouse model information and the prediction information of the phenotype caused by the gene mutation were obtained from the MGI website. The website addresses used are as follows:

[0144] NCBI:

[0145] https: / / www.ncbi.nlm.nih.gov / gene / 13511

[0146] Ensemble:

[0147] http: / / asia.ensembl.org / Mus_musculus / Gene / Summary?g

[0148] MGI:

[0149] http: / / www.informatics.jax.org / allele / summary?markerId

[0150] 1.3 Methods for constructing gene-edited mice

[0151] 1.3.1 Vector construction

[0152] Using the CRISPOR website, sgRNA was designed, constructed and in vitro transcribed according to the design plan. At the same time, the donor vector was constructed and the correctness of the vector sequence was verified by sequencing.

[0153] CRISPOR: http: / / crispor.tefor.net /

[0154] 1.3.2 Embryo microinjection

[0155] Cas9, sgRNA and Donor vector samples were microinjected into fertilized eggs of mice with C57BL / 6 background; the fertilized eggs that survived the injection were transplanted into pseudo-pregnant female mice and waited for them to become pregnant and give birth.

[0156] Table 2-1-1 Gene Editing Mouse sgRNA

[0157]

[0158] 1.3.3 Identification of F0 mice

[0159] The F0 generation mouse pups born to the recipient mice were tail-clipped and toe-clipped at 5-7 days old and numbered. Genomic DNA was extracted for PCR and sequencing to determine the genotype.

[0160] 1.3.3.1 Method for DNA extraction from mouse tissues:

[0161] 1) Reagents and consumables:

[0162] Lysis buffer: TailDigestionBuffer (500ml): 1M TrisHCl (PH = 8.0) 25ml, 0.5M EDTA 100ml, 5M NaCl 10ml, 20% SDS 25ml, MillQ 340ml, containing proteinase K (abbreviated as PK, 10mg / ml); 100% ethanol; 70% ethanol; TEBuffer (500ml): 1M Tris-HCl (PH = 8.0) 5ml, 0.5M EDTA (PH = 8.0) 1ml, MillQ 494ml, phenol and chloroform 1:1 mixed solution (stored at 4℃)

[0163] 2) Take the mouse tail (no longer than 0.3 cm) (the same applies to toes) and place it in a 1.5 ml EP tube;

[0164] 3) Prepare lysis buffer: add 5ul proteinase K to every 500ul lysis buffer, and dispense 500ul into each EP tube containing the tail;

[0165] 4) Place the EP tube with lysis solution on the EP tube rack and wrap it with plastic wrap and place it on a 55°C metal heating block (use plastic wrap to prevent the EP tube from opening due to heat and reducing the lysis solution) for overnight digestion;

[0166] 5) The next day, shake the EP tube vigorously to make the digested tissue uniform;

[0167] 6) Add an equal volume of phenol / chloroform mixed solution (500u1) to each tube;

[0168] 7) Mix vigorously and centrifuge at 12000 rpm for 15 min;

[0169] 8) Use a 1000ul pipette to draw about 300ul of supernatant from the centrifuged EP tube into a new EP tube and mark the number clearly. When drawing the supernatant, be careful not to draw the middle layer or the lower layer;

[0170] 9) Add 100% ethanol twice the volume of the supernatant (400ul) to each tube, mix by inverting, white filamentous DNA should be visible, and centrifuge at 12000rpm for 10min;

[0171] 10) Discard the supernatant, add 800ul70% ethanol, and centrifuge at 12000rpm for 5min;

[0172] 11) Discard the supernatant, carefully absorb the residual ethanol, and dry it (for more than 30 minutes) until there is no alcohol smell;

[0173] 12) Add 100ul TE solution to dissolve the DNA. The solution can be dissolved at 55℃ or 37℃ and stored at -20℃ for a long time.

[0174] 1.3.3.2 Take the extracted mouse tissue DNA and send it to the first generation sequencing to obtain the gene sequence to confirm whether the editing is successful.

[0175] 1.3.4 F0 mouse breeding and F1 mouse identification

[0176] After the positive F0 generation mice reached sexual maturity, they were mated with wild-type C57BL / 6 background mice; the tails of the F1 generation mice born were cut and the toes were numbered on days 5-7, and genomic DNA was extracted for PCR and sequencing identification;

[0177] Table 2-1-2 PCR primer information for identification of gene-edited mice

[0178]

[0179] Table 2-1-3 PCR conditions

[0180]

[0181] Table 2-1-4 PCR program

[0182]

[0183] 1) The mice obtained by targeting with the CRISPR / Cas9 system are affected by the sgRNA cutting efficiency and donor recombination efficiency, and multiple genotypes coexist in the F0 generation;

[0184] 2) After backcrossing positive F0 mice with wild-type background mice, the resulting offspring will have a single genotype;

[0185] 3) F0 generation mice cannot mate with each other;

[0186] After obtaining the F1 mice, their genotypes were identified using the same identification system as that of the F0 mice.

[0187] Mouse genotype PCR identification strategy Figure 4 .

[0188] 1.3.5 Mouse housing conditions

[0189] 1) The mice used in this experiment were raised in an SPF animal room.

[0190] 2) The temperature in the breeding room is maintained at 22-28℃, the relative humidity is maintained at 40%-60%, the light and dark alternation time is 12 / 12, and the noise is less than 60dB

[0191] 3) Feed: Sterile complete nutritional granules sterilized by 60Co gamma rays

[0192] 4) Drinking water: Bottled pure water is sterilized under high pressure and the water is changed once a week to ensure continuous drinking water supply

[0193] 5) Bedding: Use autoclaved mixed sawdust, change once a week

[0194] 6) Daily registration and observation: register various environmental data and mouse conditions every day, and closely observe the mice's diet, activities, and overall condition;

[0195] 7) The tails and toes of the pups can be docked and numbered 5-7 days after birth; ear tags and cage separation can be performed on the 19-21st day after birth. All operations must be performed in a clean bench in accordance with sterile procedures.

[0196] 2. Results

[0197] 2.1 Gene information of HIF-1α and mutation sites and design of mutant mouse sites;

[0198] 2.1.1HIF-1α gene information;

[0199] According to the data provided by NCBI, the HIF-1α gene is located on chromosome 14. Both human and mouse HIF-1α have an coded transcript, and the corresponding protein transcript numbers are NP_001834.1 and NP_031609.5, respectively. After comparison, the homology between the two is 95.3%. The amino acid at position 582 of human is proline, and the target mutation site of the mouse HIF-1α gene is also located at amino acid position 582, so the corresponding gene-edited mouse is designed at this site.

[0200] 2.1.2 Mutation sites in HIF-1α mutant mice

[0201] According to the data provided by the Ensembl website, HIF-1α has two available transcripts. Among them, the transcript numbered HIF-1α-185 (ENSMUST00000059767.15) has the highest score. This transcript encodes 13 exons, the transcript is 15766bp long, and encodes 1222 amino acids.

[0202] HE staining at 36 weeks Figure 5 .

[0203] Giemsa staining at 36 weeks Figure 6 .

[0204] 36 weeks CD34 Figure 7 .

[0205] PCR at 36 weeks Figure 8 .

[0206] 36 weeks WB Fig. 9 .

[0207] The following are two specific examples based on the above-mentioned method for establishing a mouse gastric cancer model:

[0208] Example 1: Establishment of a mouse gastric cancer model based on HIF-1α gene editing

[0209] 1.1 Selection of experimental mice

[0210] C57BL / 6 background mice were selected, and mice of 5 weeks old and weighing about 20g were selected as experimental subjects. Cas9, sgRNA and Donor vector were injected into the fertilized eggs of C57BL / 6 mice by microinjection, and gene-edited F0 generation mice were successfully generated.

[0211] 1.2 Gene editing and identification

[0212] The HIF-1α gene sequence and its protein domain information were obtained from the NCBI and Ensembl databases, and the CRISPR / Cas9 system was designed to cut the HIF-1α gene in the mouse genome. The tails of the mice were cut at 5-7 days after birth, and the genotypes of the F0 generation mice were identified by PCR and sequencing to confirm whether the gene mutation was successfully introduced.

[0213] 1.3 Breeding and F1 generation identification

[0214] Positive F0 mice were selected and mated with wild-type C57BL / 6 mice. The tails of F1 mice were cut 5-7 days after birth to extract genomic DNA, and the genotype was identified by the same method as the F0 generation to verify whether the F1 mice inherited the HIF-1α mutation. The results showed that some F1 mice successfully acquired the mutant genotype and showed gastric cancer pathological characteristics associated with HIF-1α.

[0215] 1.4 Observation and research of mouse gastric cancer model

[0216] The F1 generation mice were kept in SPF animal rooms, and their diet, activity, weight changes, and gastric lesions were regularly observed. Through tissue sections and pathological examinations, it was found that the mutant mice gradually showed characteristics of gastric cancer, such as thickening of the gastric mucosa and abnormal cell proliferation, proving that the model successfully simulated the occurrence mechanism of human gastric cancer.

[0217] Example 2: Establishment of a HIF-1α mutant mouse gastric cancer model based on CRISPR / Cas9

[0218] 2.1 Experimental design and fertilized egg injection

[0219] The mutation model information of HIF-1α gene in mice was obtained from MGI database, sgRNA was designed and Donor vector was constructed. Cas9, sgRNA and Donor vector were injected into fertilized eggs of C57BL / 6 background mice by microinjection, transplanted into pseudo-pregnant female mice, and waited for them to give birth.

[0220] 2.2 Identification of F0 generation mice

[0221] After the F0 generation mice were born, their tails were cut off 5-7 days later, and the mouse tissue DNA was extracted using DNA extraction reagents and sent to the sequencing center for first-generation sequencing. The results showed that some F0 generation mice successfully acquired HIF-1α gene mutations, indicating that the gene editing operation was successful.

[0222] 2.3 F1 generation breeding and genotype confirmation

[0223] Positive F0 mice were selected and mated with wild-type C57BL / 6 mice. After the F1 mice were born, their genotypes were identified by PCR and sequencing to ensure that the mutant gene was successfully transmitted to the F1 mice. Among the F1 mice, some individuals showed a gastric cancer phenotype associated with HIF-1α mutation.

[0224] 2.4 Further studies on the mouse gastric cancer model

[0225] The F1 generation mice were raised in a standardized environment, and regular gastric tissue sections and tumor marker tests were performed to further analyze how HIF-1α gene mutations affect the occurrence and development of gastric cancer. The study showed that these mice gradually developed gastric cancer, verifying the reliability of the gastric cancer model and providing an experimental basis for subsequent gastric cancer drug screening and treatment research.

[0226] It should be noted that the embodiments of the present invention can be implemented by hardware, software, or a combination of software and hardware. The hardware part can be implemented using dedicated logic; the software part can be stored in a memory and executed by an appropriate instruction execution system, such as a microprocessor or dedicated design hardware. It can be understood by a person of ordinary skill in the art that the above-mentioned devices and methods can be implemented using computer executable instructions and / or contained in a processor control code, such as a carrier medium such as a disk, CD or DVD-ROM, a programmable memory such as a read-only memory (firmware), or a data carrier such as an optical or electronic signal carrier. Such code is provided on the carrier medium. The device and its modules of the present invention can be implemented by hardware circuits such as very large-scale integrated circuits or gate arrays, semiconductors such as logic chips, transistors, etc., or programmable hardware devices such as field programmable gate arrays, programmable logic devices, etc., can also be implemented by software executed by various types of processors, and can also be implemented by a combination of the above-mentioned hardware circuits and software, such as firmware.

[0227] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for establishing a mouse gastric cancer model, characterized in that: The following steps are involved: Step 1, selection of experimental mice: mice with C57BL / 6 background and their fertilized eggs were selected; Step 2: Obtaining genetic information: Obtain human and mouse HIF-1α gene sequence, protein sequence and domain information from NCBI and Ensembl websites, perform homology comparisons, and obtain HIF-1α gene transcript information; obtain current HIF-1α mutant mouse model information and prediction information on the phenotype caused by the gene mutation from the MGI website; Step 3, construction of gene-edited mice; Vector construction: According to the design plan, design, construct and in vitro transcribe sgRNA, construct the source recombinant vector, and verify the correctness of the vector sequence by sequencing; Embryo microinjection: Cas9, sgRNA and Donor vector samples were microinjected into fertilized eggs of mice with C57BL / 6 background; fertilized eggs that survived the injection were transplanted into pseudo-pregnant female mice and waited for them to become pregnant and give birth; Identification of F0 mice: The F0 generation mice pups born to the recipient mice were tail-clipped and toe-clipped at 5-7 days old, and their genomic DNA was extracted for PCR and sequencing to determine their genotypes; F0 mouse breeding and F1 mouse identification; mouse breeding conditions.

2. The method for establishing a mouse gastric cancer model according to claim 1, characterized in that: The identification method of the F0 mice is as follows: (1) DNA extraction from mouse tissues; (2) Take the extracted mouse tissue DNA and send it to the first-generation sequencing to obtain the gene sequence to confirm whether the editing is successful.

3. The method for establishing a mouse gastric cancer model according to claim 2, characterized in that: The mouse tissue DNA extraction method: 1) Reagents and consumables: Lysis buffer: TailDigestionBuffer (500ml): 1M TrisHCl (PH=8.0) 25ml, 0.5M EDTA 100ml, 5M NaCl 10ml, 20% SDS 25ml, MillQ 340ml, containing proteinase K (PK, 10mg / ml); 100% ethanol; 70% ethanol; TEBuffer (500ml): 1M Tris-HCl (PH=8.0) 5ml, 0.5M EDTA (PH=8.0) 1ml, MillQ 494ml, phenol and chloroform 1:1 mixed solution; 2) Take the mouse tail and put it into a 1.5 ml EP tube; 3) Prepare lysis buffer: add 5ul proteinase K to every 500ul lysis buffer, and dispense 500ul into each EP tube containing the tail; 4) Place the EP tube with lysis solution on the EP tube rack, wrap it with plastic wrap and place it on a metal heating block at 55°C for overnight digestion; 5) The next day, shake the EP tube vigorously to make the digested tissue uniform; 6) Add 500ul of phenol / chloroform mixed solution of equal volume to each tube; 7) Mix vigorously and centrifuge at 12000 rpm for 15 min; 8) Use a 1000ul pipette to draw about 300ul of supernatant from the centrifuged EP tube into a new EP tube and mark the number clearly. When drawing the supernatant, be careful not to draw the middle layer or the lower layer; 9) Add 100% ethanol twice the volume of the supernatant (400ul) to each tube, mix by inverting, white filamentous DNA should be visible, and centrifuge at 12000rpm for 10min; 10) Discard the supernatant, add 800ul70% ethanol, and centrifuge at 12000rpm for 5min; 11) Discard the supernatant, carefully absorb the residual ethanol, and dry it until there is no alcohol smell; 12) Add 100ul TE solution to dissolve the DNA. The solution can be dissolved at 55℃ or 37℃ and stored at -20℃ for a long time.

4. The method for establishing a mouse gastric cancer model according to claim 3, characterized in that: The 1:1 mixed solution of phenol and chloroform was stored at 4°C; The length of the mouse tail does not exceed 0.3 cm; Drying time is more than 30 minutes.

5. The method for establishing a mouse gastric cancer model according to claim 1, characterized in that: The F0 mouse breeding and F1 mouse identification: After the positive F0 generation mice reached sexual maturity, they were mated with wild-type C57BL / 6 background mice; the tails of the F1 generation mice born were cut and the toes were numbered on days 5-7, and genomic DNA was extracted for PCR and sequencing identification; 1) The mice obtained by targeting with the CRISPR / Cas9 system are affected by the sgRNA cutting efficiency and donor recombination efficiency, and multiple genotypes coexist in the F0 generation; 2) After backcrossing positive F0 mice with wild-type background mice, the resulting offspring will have a single genotype; 3) F0 generation mice cannot mate with each other; After obtaining the F1 mice, their genotypes were identified using the same identification system as that of the F0 mice.

6. The method for establishing a mouse gastric cancer model according to claim 1, characterized in that: The mouse breeding conditions: 1) The mice used in this experiment were raised in an SPF animal room. 2) The temperature in the breeding room is maintained at 22-28°C, the relative humidity is maintained at 40%-60%, and the light-dark cycle is 12 / 12; Noise<60dB 3) Feed: Sterile complete nutritional granules sterilized by 60Co gamma rays 4) Drinking water: Bottled pure water is sterilized under high pressure and the water is changed once a week to ensure continuous drinking water supply 5) Bedding: Use autoclaved mixed sawdust, change once a week 6) Daily registration and observation: register various environmental data and mouse conditions every day, and closely observe the mice's diet, activities, and overall condition; 7) The tails and toes of the pups can be docked and numbered 5-7 days after birth; ear tags and cage separation can be performed on the 19-21st day after birth. All operations must be performed in a clean bench in accordance with sterile procedures.

7. A system for establishing a mouse gastric cancer model for implementing the method for establishing a mouse gastric cancer model according to any one of claims 1 to 6, characterized in that: The mouse gastric cancer model establishment system comprises: Selection module, used to select mice with C57BL / 6 background and their fertilized eggs; Gene information acquisition module, used to obtain human and mouse HIF-1α gene sequence, protein sequence and domain information from NCBI and Ensembl websites, and perform homology comparison to obtain transcript information of HIF-1α gene; obtain current HIF-1α mutant mouse model information and prediction information on the phenotype caused by the gene mutation from the MGI website; Building blocks for gene-edited mouse construction.

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