Construction method and application of HRAS humanized mouse for carcinogenicity evaluation
By constructing a mouse model carrying human HRAS genes that induce cancer intron mutations, the problem of long trial cycle and high cost in existing carcinogenic evaluation techniques was solved, and efficient and economical carcinogenic evaluation was achieved, and the incidence of spontaneous tumors reached 95%.
Patent Information
- Application Number
- CN202411936582.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-12-26
- Filing Date
- 2024-12-26
- Publication Date
- 2025-06-27
AI Technical Summary
Among the existing carcinogenicity evaluation techniques, the use of rats to conduct experiments has the disadvantages of long cycles, high costs, and low probability of spontaneous tumors, and it is difficult to complete carcinogenicity evaluation efficiently and economically.
By constructing mice carrying human HRAS genes that only retain cancer-related intron mutations, prokaryotic injection technology was used to insert HRAS gene fragments into the mouse genome to form an HRAS humanized mouse model for oncogenicity evaluation.
This method significantly improves the incidence of spontaneous tumors to 95%, shortens the trial cycle of carcinogenic evaluation, reduces the cost of trials, and reduces the number of animals used.
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Figure CN120210285A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and particularly to a method for constructing an HRAS humanized mouse for carcinogenicity evaluation and its application. Background Art
[0002] Carcinogenicity evaluation is an important part of non-clinical safety evaluation in new drug research and development. It can identify the potential carcinogenic effects of drugs. Selecting a suitable animal model can effectively expose the potential carcinogenicity of drugs and greatly affect the data and results of drug carcinogenicity evaluation.
[0003] To meet the animal model requirements for drug carcinogenicity evaluation, there are already transgenic animal models on the market (such as p53+ / - deletion model, TgAC model, TgHras2 model, XPA deletion model, etc.). By introducing proto-oncogenes or knocking out tumor suppressor genes, these transgenic animals are made more sensitive to carcinogenic factors, thus compensating for the deficiencies of using rats in carcinogenicity evaluation tests.
[0004] The rasH2 transgenic mouse developed by CIEA in Japan has introduced a modified proto-oncogene HRAS, increasing the sensitivity of the transgenic mouse to external stimulating factors and making it more likely to develop spontaneous malignant cancers. Literature reports that under the stimulation of the positive carcinogen N-methyl-N-nitrosourea (MNU), the rasH2 mouse developed spontaneous in-situ malignant lymphoma, gastric cancer, and skin cancer, and the incidence of these cancer types was between 30% and 50%. At the same time, there were also phenotypes such as spontaneous lung cancer and ovarian cancer, which confirmed the feasibility of using the rasH2 mouse in carcinogenicity evaluation tests. Compared with rats, using the rasH2 mouse in carcinogenicity evaluation tests has the advantages of shortening the test cycle (half a year), reducing the number of animals used by 50%, reducing test costs, and increasing the probability of spontaneous cancer. Therefore, it has important application value in drug carcinogenicity evaluation. However, it should be noted that currently, using the rasH2 mouse in carcinogenicity evaluation tests still consumes a large amount of manpower and material resources. The in-vivo test cycle is half a year, at least 200 mice are required for a formal carcinogenicity evaluation test, and the workload of pathological sectioning in the later stage is large, which is closely related to the incidence of spontaneous tumors. Based on the rasH2 mouse, further increasing the incidence of spontaneous tumors is one of the effective methods to shorten the carcinogenicity evaluation test cycle and reduce the number of test animals used.
[0005] The HRAS gene used in rasH2 transgenic mice contains the promoter region derived from the HRAS gene of patients with malignant melanoma and the intron mutation sites in the HRAS proto-oncogene of bladder adenocarcinoma, and is inserted into the mouse chromosome region 15E3 in a tandem sequence of three copies by random transgenesis. The carcinogenic characteristics of rasH2 transgenic mice are closely related to the selection of the HRAS gene fragment and the transgene insertion site. By selecting different HRAS gene fragments (the promoter region of the HRAS gene of normal people) and knocking them into mice by random transgenesis, HRAS transgenic animal models with completely different insertion sites, copy numbers, and gene sequences can be obtained, thereby affecting the carcinogenic characteristics of the animal model to different drugs, obtaining more sensitive animal models with a higher spontaneous cancer probability, and thus improving and perfecting the application of HRAS transgenic animal models in the field of carcinogenicity evaluation.
[0006] However, traditional carcinogenicity evaluation tests are mostly carried out using rats. Due to the complex factors inducing cancer and the low probability, using rats for carcinogenicity evaluation has the disadvantages of a long evaluation period (2 years), high test costs, a large number of animals in each group, and a low probability of spontaneous tumors. Summary of the Invention
[0007] (1) Technical problems to be solved
[0008] In view of the deficiencies of the prior art, the present invention provides a method for constructing an HRAS humanized mouse for carcinogenicity evaluation and its application. Using a human HRAS gene carrying only the intron mutations related to cancer induction and transferring it into mouse embryos, the obtained HRAS humanized mouse is more efficient for carcinogenic verification, and has a high incidence rate. It can be quickly discovered during the verification process, achieving the purpose of reducing the verification period, reducing test costs, and having a high probability of spontaneous tumors, thus solving the problems raised in the background technology.
[0009] (2) Technical solutions
[0010] To achieve the above object, the present invention provides the following technical solutions: A method for constructing an HRAS humanized mouse for carcinogenicity evaluation, comprising the following steps:
[0011] Construction of Tg-HRAS mice: Inject the constructed vector into mouse fertilized eggs by pronuclear injection, and randomly insert the human HRAS gene fragment into the mouse genome, including the HRAS gene regulatory sequence and the expression sequence. The human HRAS gene sequence fragment is as shown in SEQ ID NO.1;
[0012] Construction of the PMD18-T backbone vector: Using the PMD18-T vector as a template, perform high-fidelity PCR amplification to obtain a 2.7 kb linearized PMD18-T vector, and recover the gel as the backbone for the SLIC reaction;
[0013] Preparation of HRAS fragments: Human HRAS was prepared in three segments. Fragments were prepared using BAC as a template, and the corresponding fragments were obtained by PCR amplification with high-fidelity enzymes. The gel-purified products were used as fragments for the SLIC reaction.
[0014] SLIC ligation: The PMD18-T vector, Hras-H1, Hras-H2, and Hras-H3 sequences were ligated by SLIC and identified by PCR, restriction enzyme digestion, and sequencing.
[0015] Preparation of transplantation products: Digested with EcoRV to obtain a 6488 + 2698 bp band, and the 6488 bp product was recovered for transplantation.
[0016] Obtaining positive mice by transplantation injection: The transplantation products were injected into fertilized eggs and transplanted into pseudopregnant mice to obtain positive F0. The genotypes of the offspring born to pseudopregnant mice were identified and sequenced, and positive F0 mice with the correct human fragment successfully inserted were screened.
[0017] Establishment of the founder mouse line: The obtained F0 was mated with background mice to obtain F1, and F1 was mated with background mice again to obtain F2. Each generation was mated with background mice to obtain positive offspring, and the copy number was monitored by QPCR. Detection was continued until the N5 generation, and the mouse copy number was maintained at 4, indicating successful establishment of the line.
[0018] Preferably, the successfully identified vector in the SLIC ligation is named Tg-HRAS-donor.
[0019] A vector obtained by the method for constructing an HRAS humanized mouse for carcinogenicity evaluation as described above.
[0020] Application of the method for constructing an HRAS humanized mouse for carcinogenicity evaluation as described above, including verification of human HRAS mRNA expression:
[0021] Take different tissues of positive mice and background mice, extract RNA using the Trizol method, obtain samples by reverse transcription, and perform qRT-PCR identification.
[0022] Application of the method for constructing an HRAS humanized mouse for carcinogenicity evaluation as described above, including verification of human HRAS protein expression:
[0023] Mate BALB / c female mice with B6-hHRAS male mice to obtain BALB / c; B6 and BALB / c; B6-Tg. Take the lung, liver, skin, spleen, forestomach, and thymus tissues of positive mice and background mice, and detect HRAS protein using Western blot technology.
[0024] Application of a method for constructing HRAS humanized mice for carcinogenicity evaluation as described above, including verification of spontaneous tumor formation in mice.
[0025] Application of a method for constructing HRAS humanized mice for carcinogenicity evaluation as described above, including verification of carcinogenesis induced by carcinogen MNU in mice.
[0026] Application of a method for constructing HRAS humanized mice for carcinogenicity evaluation as described above in drug carcinogenicity evaluation.
[0027] Application of a method for constructing HRAS humanized mice for carcinogenicity evaluation as described above in the preparation of a mouse model for carcinogenicity evaluation.
[0028] In the application in drug carcinogenicity evaluation or in the preparation of a mouse model for carcinogenicity evaluation as described above, the cancers include lung adenocarcinoma, lymphoma, skin papilloma, keratoacanthoma, cutaneous squamous cell carcinoma, gastric squamous cell tumor / carcinoma, Harderian adenoma or hemangioma.
[0029] (III) Beneficial effects
[0030] Compared with the prior art, the present invention has the following beneficial effects:
[0031] Using human HRAS genes carrying only intron mutations related to cancer induction to construct HRAS humanized mice, and using these mice for carcinogenic verification. From the verification experimental data, it can be seen that the verification results are more efficient, and the tumor incidence rate is as high as 95%. It can be quickly discovered during the verification process, achieving the purpose of reducing the verification cycle, reducing the test cost, and having a high probability of spontaneous tumors. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 is the identification electrophoresis diagram of the 5'-end ( Figure 1A ) and 3'-end ( Figure 1B ) of the present invention. In the figure, WT is B6 genomic DNA; N is a blank control, a control without a template; P is a positive control, a positive plasmid;
[0033] TRANS2K PLUS II bands:
[0034] 8000bp\5000bp\3000bp\2000bp\1000bp\750bp\500bp\250bp\100bp;
[0035] Figure 2 is the detection diagram of the relative expression level of human HRAS mRNA of the present invention;
[0036] Figure 3 is the detection of human HRAS protein expression of the present invention;
[0037] Figure 4 This is the mortality curve of the present invention. Detailed implementation mode
[0038] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0039] Embodiment 1
[0040] Referring to FIG. 1, a method for constructing a HRAS humanized mouse for carcinogenicity evaluation includes the following steps:
[0041] Construction of Tg-HRAS mice: The constructed vector is injected into mouse fertilized eggs by pronuclear injection, and the human HRAS gene fragment, including the HRAS gene regulatory sequence and the expression sequence, is randomly inserted into the mouse genome. The human HRAS gene sequence fragment is as shown in SEQ ID NO.1.
[0042] Construction of the vector and transplantation product includes the construction of the PMD18-T backbone vector, preparation of the HRAS fragment, SLIC ligation, and preparation of the transplantation product.
[0043] Construction of the PMD18-T backbone vector: Using the primers in Table 1, with the PMD18-T vector (TaKaRa Cat#D101A) as the template, a 2.7 kb linearized PMD18-T vector is obtained by high-fidelity PCR amplification, and gel recovery is used as the backbone for the SLIC reaction.
[0044] Preparation of the HRAS fragment: The human HRAS is prepared in three segments (Hras-H1, Hras-H2, and Hras-H3). Fragments are prepared using BAC as the template, and the corresponding fragments are obtained by high-fidelity enzyme PCR amplification using the primers in Table 1. Gel recovery is used as the fragment for the SLIC reaction.
[0045] Table 1 SLIC fragment amplification primers
[0046]
[0047]
[0048] SLIC ligation: The PMD18-T vector, Hras-H1, Hras-H2, and Hras-H3 sequences are ligated by SLIC, and the successfully identified vector is named Tg-HRAS-donor.
[0049] Identification was carried out by PCR identification, restriction enzyme digestion and sequencing identification methods. The specific identification primers and protocols are shown in Tables 2, 3 and 4.
[0050] Preparation of transplantation products: Digest with EcoRV to obtain 6488 + 2698 bp bands, and recover the 6488 bp product for transplantation.
[0051] Table 2 Identification primers for vector construction
[0052]
[0053] Table 3 Restriction enzyme digestion identification protocol
[0054]
[0055] Table 4 Sequencing protocol
[0056]
[0057]
[0058] Obtaining positive mice by transplantation injection:
[0059] Inject the transplantation product into the fertilized egg and transplant it into the pseudopregnant mouse;
[0060] Obtain positive F0, perform genotype identification and sequencing on the offspring born to the pseudopregnant mice. The identification primers are shown in Table 5, and the sequencing primers are shown in Table 6;
[0061] Screen positive F0 mice with the correct human-derived fragment successfully inserted. The PCR experimental results of F0 mice are shown in Figure 1, and the obtained mouse numbers are as follows: 123, 124, 148#.
[0062] Table 5 F0 identification protocol
[0063]
[0064] Table 6 F0 sequencing protocol
[0065]
[0066] Establishing the founder mouse line: Mate the obtained F0 with the background mouse to obtain F1, and then mate F1 with the background mouse to obtain F2. Each generation is mated with the background mouse to obtain positive offspring, and the copy number is monitored by QPCR, continuously detecting until the N5 generation. The mouse copy number is maintained at 4, and the line establishment is successful.
[0067] The QPCR detection method is as follows:
[0068]
[0069] Example 2
[0070] Figures 2-4 It includes the verification of human HRAS mRNA expression, the verification of human HRAS protein expression, the verification of spontaneous tumorigenesis in mice, and the verification of carcinogenesis induced by the carcinogen MNU in mice.
[0071] Verification of human HRAS mRNA expression: Different tissues of positive mice and background mice were taken, RNA was extracted using the Trizol method, samples were obtained by reverse transcription, and qRT-PCR identification was performed. The identification primers are shown in Table 7 below, and the identification results are shown in Figure 2 .
[0072] Table 7 Q-PCR identification scheme
[0073]
[0074] Verification of human HRAS protein expression:
[0075] BALB / c female mice were mated with B6-hHRAS male mice to obtain BALB / c; B6 and BALB / c; B6-Tg(HRAS). Lung, liver, skin, spleen, forestomach, and thymus tissues of positive mice and background mice were taken, and Western blot technology was used to detect HRAS protein. The detection results are shown in Figure 3 . It can be seen from the figure that human HRAS protein can be successfully expressed in B6-hHRAS mice.
[0076] Verification of spontaneous tumorigenesis in mice:
[0077] BALB / c female mice were mated with B6-hHRAS male mice to obtain BALB / c; B6 and BALB / c; B6-Tg(HRAS). 50 pairs of BALB / c; B6-Tg(HRAS) mice, 63 female and 57 male BALB / c; B6 mice were taken. The mice were observed for 52 weeks, gross dissections were performed on the mice, abnormal tissues were taken for HE staining to detect tissue abnormalities, and the number and types of spontaneous tumor mice were counted. The results are shown in Table 8.
[0078] Compared with the background mice BALB / c; B6, male BALB / c; B6-Tg(hHRAS) mice will develop lung adenocarcinoma, lymphoma, skin papilloma, keratoacanthoma, cutaneous squamous cell carcinoma, and gastric squamous cell tumor / carcinoma, and female mice will develop lung adenocarcinoma, Harderian adenoma, and hemangioma.
[0079] Table 8 Types and probabilities of spontaneous tumors in mice
[0080]
[0081]
[0082] Verification of carcinogenesis induced by carcinogen MNU in mice:
[0083] Forty pairs of 6-9-week-old BALB / c; B6, BALB / c; B6-Tg(hHRAS) were selected and randomly divided into 4 groups according to the groups in Table 9. The BALB / c; B6 normal saline group (15 animals / sex / group), the BALB / c; B6 MNU group (15 animals / sex / group), the BALB / c; B6-Tg(hHRAS) normal saline group (15 animals / sex / group), and the BALB / c; B6 MNU group (15 animals / sex / group). The normal saline groups were administered by gavage once a day for a total of 26 weeks; the MNU groups were given by intraperitoneal injection, administered once on D1. During the experiment, the growth status and clinical symptoms of the animals were monitored. After 26 weeks of monitoring, all animals were subjected to systematic dissection and gross observation, and the tissues of the animals were collected: adrenal gland, aorta, bone and bone marrow, sternum and femur, brain, coagulating gland, clitoral gland, epididymis, esophagus, eyeball with optic nerve, Harderian gland, heart, tibiofemoral joint, kidney, lacrimal gland, large intestine (cecum, colon, rectum), larynx, liver, gallbladder, lung with main bronchus, lymph nodes (submandibular lymph nodes, mesenteric lymph nodes, inguinal lymph nodes), mammary gland, sciatic nerve, nose (3 sections), ovary with fallopian tube, Peyer's patches, pancreas, pharynx, preputial gland, prostate, pituitary gland, salivary glands (3 pairs), seminal vesicle, skeletal muscle (biceps femoris), skin (around the mammary gland), small intestine (duodenum, jejunum, ileum), spinal cord (cervical, thoracic, lumbar segments), spleen, stomach (glandular area and non-glandular area), testis, thymus, thyroid gland (including parathyroid gland), tongue, trachea, bladder, uterus with cervix, vagina.
[0084] Table 9 Drug administration grouping
[0085]
[0086] Conventional histological processing was carried out as needed, including paraffin embedding, sectioning, spreading, HE staining, etc. All tissue sections of the animals were examined under the microscope. The death situation and tumor occurrence of the mice were counted. The death curve is shown in Figure 4 . It can be seen from Figure 4 that after exposure to the carcinogen MNU, both BALB / c; B6 and BALB / c; B6-Tg(hHRAS) mice died, but the death of BALB / c; B6-Tg(hHRAS) mice was more severe.
[0087] The evaluation of the carcinogenic characteristics of BALB / c; B6-Tg(hHRAS) mice by MNU is shown in Table 10. WT.Hras mice are the littermate negative controls of Tg.Hras mice and do not contain the human HRAS gene. As can be seen from the results in Table 10, compared with WT.Hras mice, after induction with MNU, Tg.Hras mice had higher incidences of lymphoma, skin papilloma, and gastric squamous cell papilloma. This indicates that the inserted human HRAS gene in Tg.Hras mice significantly promoted the generation of different types of spontaneous tumors. The carcinogen MNU induced lymphoma and papilloma / carcinoma in the stomach in 95% of the mice. Compared with the results in the literature (Usui, T, Mutai, M and Hisada, S, et al., 2001), the incidences of lymphoma and papilloma / carcinoma in the stomach induced by the carcinogen MNU in mice were significantly higher than those reported for rasH2 mice in the literature. It can be seen that BALB / c; B6-Tg(hHRAS) mice are more sensitive to the induction of the carcinogen MNU than rasH2 mice and have a higher incidence of spontaneous tumors.
[0088] Table 10 Evaluation of the Carcinogenic Characteristics of MNU
[0089]
[0090]
[0091] In summary, the carcinogen can induce tumors in BALB / c; B6-Tg(hHRAS), and BALB / c;
[0092] B6-Tg(hHRAS) can be used to evaluate the carcinogenic characteristics of carcinogens. Compared with the rasH2 mice reported in the existing literature, BALB / c; B6-Tg(hHRAS) is more sensitive to carcinogens and has a higher probability of spontaneous tumor occurrence, which is helpful for carrying out carcinogenic evaluation tests.
[0093] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for constructing HRAS humanized mice for carcinogenicity evaluation, characterized in that: The following steps are involved: Construction of Tg-HRAS mice: The constructed vector is injected into mouse fertilized eggs by pronuclear injection, and a human HRAS gene fragment, including a HRAS gene regulatory sequence and an expression sequence, is randomly inserted into the genome of the mouse. The human HRAS gene sequence fragment is shown in SEQ ID NO.1; Construction of PMD18-T backbone vector: Using PMD18-T vector as template, high-fidelity PCR amplification was performed to obtain a 2.7 kb linearized PMD18-T vector, which was recovered by gel as the backbone for SLIC reaction; Preparation of HRAS fragments: Human HRAS was divided into three segments. The fragments were prepared using BAC as a template, and the corresponding fragments were obtained by high-fidelity enzyme PCR amplification. The fragments were recovered by gel as the fragments for SLIC reaction; SLIC ligation: PMD18-T vector, Hras-H1, Hras-H2 and Hras-H3 sequences were SLIC-ligated and identified by PCR, restriction digestion and sequencing; Preparation of transplantation products: digest with EcoRV to obtain a 6488+2698bp band, and recover the 6488bp product for transplantation; Transplantation injection to obtain positive mice: Inject the transplanted product into fertilized eggs, transplant into pseudo-pregnant mice, obtain positive F0, identify and sequence the genotypes of pseudo-pregnant mice, and screen for positive F0 mice with successful insertion of the correct human fragment; Founder mouse line establishment: The obtained F0 is bred with background mice to obtain F1, and F1 is bred with background mice to obtain F2. Each generation is bred with background mice to obtain positive offspring, and the copy number is monitored by QPCR. The detection is continued until the N5 generation. The mouse copy number is maintained at 4, and the line is successfully established.
2. The method for constructing a HRAS humanized mouse for carcinogenicity evaluation according to claim 1, characterized in that: The vector successfully identified in the SLIC connection was named Tg-HRAS-donor.
3. A vector obtained according to the method for constructing HRAS humanized mice for carcinogenicity evaluation according to claim 1 or 2.
4. The use of a method for constructing a HRAS humanized mouse for carcinogenicity evaluation according to any one of claims 1 to 2, characterized in that: Including human HRAS mRNA expression verification: Different tissues of positive mice and background mice were taken, RNA was extracted using the Trizol method, and samples were obtained using the reverse transcription method for qRT-PCR identification.
5. The use of the method for constructing a HRAS humanized mouse for carcinogenicity evaluation according to any one of claims 1 to 2, characterized in that: Including human HRAS protein expression verification: BALB / c female mice were bred with B6-hHRAS male mice to obtain BALB / c;B6 and BALB / c;B6-Tg. The lungs, livers, skins, spleens, forestomachs, and thymus tissues of positive mice and background mice were obtained, and the HRAS protein was detected using Western blot technology.
6. The use of the method for constructing a HRAS humanized mouse for carcinogenicity evaluation according to any one of claims 1 to 2, characterized in that: Including spontaneous tumorigenesis in mice.
7. The use of the method for constructing a HRAS humanized mouse for carcinogenicity evaluation according to any one of claims 1 to 2, characterized in that: Including the validation of carcinogenicity induced by carcinogen MNU in mice.
8. Use of the method for constructing a HRAS humanized mouse for carcinogenicity evaluation according to any one of claims 1 to 2 in drug carcinogenicity evaluation.
9. Use of the method for constructing a HRAS humanized mouse for carcinogenicity evaluation according to any one of claims 1 to 2 in preparing a HRAS humanized mouse model for carcinogenicity evaluation.
10. The use according to claim 8 or 9, characterized in that: The cancers include lung adenocarcinoma, lymphoma, skin papilloma, keratoacanthoma, skin squamous cell carcinoma, stomach squamous cell tumor / carcinoma, Harderian adenoma or hemangioma.