Construction method and application of mouse exogenous gene expression model based on adenovirus vector

Abstract

The invention belongs to the technical field of biology, and relates to a construction method and application of a mouse exogenous gene expression model based on an adenovirus vector. The exogenous gene is delivered through the adenovirus vector, the exogenous gene is subjected to site-specific integration to the specific site of the mouse target cell by using the double recombinase, the efficiency is high, the integration site and copy number are controllable, the safety is good, various function acquired variations can be efficiently modeled, and the method can be applied to establishment of a tumor model and research of precision medical treatment.

Description

[0001] Cross-reference to related applications This invention claims priority to Chinese Patent Application No. 202510126226.4, filed on January 27, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biotechnology, specifically relating to an adenovirus vector, its preparation method, and its application. Background Technology

[0003] Mouse models are common animal models for preclinical drug trials. Because mice (… Mus musculus With over 90% similarity to the human genome and being easy to raise and inexpensive to breed, mouse models have become a universally applicable model organism. Genetically engineered mouse models (GEMMs) are important tools for studying disease mechanisms (pathology), pharmacology, and genetics. In oncology, GEMMs can reproduce tumor development, progression, metastasis, and drug resistance. Conditional GEMMs, through the spatiotemporal specific expression of recombinases such as Cre / loxP and Flp / FRT, can achieve conditional activation of oncogenes, gene amplification, or overexpressed genes, as well as conditional knockout of tumor suppressor genes, thereby identifying tumor driver genes and elucidating the function of mutated genes. Cre / loxP has multiple variants such as Dre / rox, VCre / VloxP, and SCre / SloxP. In addition, loxP that can combine with Cre also has multiple variants such as lox66, lox71, and lox2272. Flp has multiple variants with sequence identity reaching 98%, such as FlpE and FlpO, with FlpO exhibiting higher recombination efficiency. Establishing a conditional GEMM generally requires knocking in sequences such as loxP and FRT into the gene locus to be studied, which takes a long time.

[0004] International research on animal models of diseases is currently booming. Genome-wide association studies (GWAS) and the Cancer Genome Project have identified a large number of disease-related gene mutations, gene amplifications, gene deletions, and structural variations (chromosomal translocations, etc.), the functions of most of which remain unclear. Variations that promote disease are called driver variants, and genes exhibiting driver variants are called driver genes. In revealing driver genes, mouse models can better simulate human disease processes and are far cheaper than those of large mammals and primates. Driver variants can be divided into gain-of-function variants and loss-of-function variants based on their impact on gene function. Modeling loss-of-function variants requires disrupting the original function of the gene; the current development of CRISPR knockout technology has greatly facilitated this, leading to a large number of CRISPR-based loss-of-function mouse models. Modeling gain-of-function variants is more complex and has significant gaps in understanding.

[0005] Current technology for modeling gain-of-function variants (GEMMs) utilizes genetically engineered mouse models. Numerous GEMMs exist in diseases such as tumors, including gene amplification (copy number amplification), gain-of-function point mutations, specific structural variations (gene fusion, chromatin flipping, etc.), and increased gene expression. Establishing disease mouse models of GEMMs helps elucidate the roles of these gene variations and discover new oncogenes, drug targets, and diagnostic targets. Traditional GEMMs first integrate genes such as KRAS-G12D mutants, EGFR-T790M mutants, and SOX2 into embryonic stem cells through homologous recombination. These embryonic stem cells are then implanted into the inner cell mass for embryo transfer, producing chimeric mice. These mice are then bred to establish GEMMs. Kras LSL-G12D , EGFR LSL-T790M , Col1a1 LSL-Sox2 Gene knock-in mouse strains are collectively referred to as exogenous genes. Traditional GEMM requires establishing single-genotype mouse strains for each gene under study, resulting in low throughput for genotype modeling; if research is required... Kras-G12D;p53 , EGFR-T790M;p53 , SOX2;Cdkn2a;Pten For complex genotypes, at least one generation of hybridization and one generation of self-crossing are required, which is time-consuming and costly. These established mouse strains typically activate exogenous genes using recombinases such as Cre and Flp to avoid embryonic lethality caused by exogenous gene overexpression. Several tissue-specific recombinase transgenic mouse strains are already commercially available, such as those expressing only in alveolar epithelial cells. Sftpc-Cre ERT2 strains can be obtained through... Kras LSL-G12D , EGFR LSL-T790M, Col1a1 LSL-Sox2 The process of hybridizing mouse strains to generate conditionally activating genotypes, thereby activating functional gains in tissues such as the lung epithelium, further increases the time cost. Tyler Jacks' lab at MIT has saved time on hybridization and purification of recombinase mouse strains by delivering recombinase via endotracheal intubation using lentivirus and adenovirus vectors, which has been widely used in lung cancer modeling. However, establishing gene knock-in mouse strains remains time-consuming and labor-intensive.

[0006] CRISPR-based homology repair can model hotspot mutations, but it is inefficient. Delivering exogenous genes using lentiviral vectors or CRISPRa-designed sgRNAs can produce a copy number increase-like effect, but in practice, we found that lentivirally integrated sequences are silenced after 3 weeks in mouse models, thus limiting this method to in vitro experiments or transient expression. Adenoviruses have strong carrying capacity, capable of carrying 7.5 kb, and have a very low probability of spontaneous embedding into the cellular genome, making them commonly used for transient expression. Transfection plasmids are easy to manipulate in vitro, but they cannot replicate in eukaryotic cells, limiting their application to transient expression. Although transposons can integrate long fragments, the insertion site is random and the copy number is uncontrollable. In Drosophila, CRISPR-mediated homology recombination can integrate plasmids to specific sites; however, its feasibility in mice has not been validated. Recombinases can induce site-specific integration of exogenous genes in cell lines, but integration based on a single FRT site is reversible. Recently, American researchers injected Cre and Flp double recombinases into the brains of fetal mice and delivered the foreign gene using electric shock. However, this method is invasive, which can cause wounds to the mice, and the electric shock operation is inconvenient, which limits the efficiency and safety of modeling.

[0007] Therefore, there is an urgent need to develop a technology for long-term, stable, and convenient modeling capabilities to acquire variation. Summary of the Invention

[0008] This invention uses an adenovirus vector to deliver exogenous genes and double recombinases, and utilizes Cre and FlpO double recombinases for site-specific integration, overcoming the shortcomings of existing technologies.

[0009] On one hand, the present invention provides an adenovirus vector, characterized in that the vector contains a foreign gene and a gene expressing a double recombinase, and is capable of site-specific integration of the foreign gene into a specific site in mouse target cells via the double recombinase.

[0010] In some embodiments, the dual recombinases are recombinase FlpO or a variant thereof and recombinase Cre or a variant thereof.

[0011] In some embodiments, the recombinase FlpO variant includes FlpE or Flp.

[0012] In some implementations, the recombinase Cre variant includes Dre or VCre.

[0013] In some embodiments, the amino acid sequence of the recombinase FlpO or a variant thereof comprises the sequence shown in SEQ ID NO:1-2 or an amino acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to it. The sequences in SEQ ID NO:1-2 are shown below: SEQ ID NO:1: FlpO MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAAELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPA WEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQ NKYLGVIIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFL SMKGLTELTNVVGNWSDKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI SEQ ID NO:2: Flp MPQFDILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMITHNGTAIKRATFMSYNTIISNSLSLDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTII PYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYL GVIIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYNKALKKNAPYSIFAIKNGPKSHIGRHLMTSFLSM KGLTELTNVVGNWSDKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI The amino acid sequence of FlpE is 99% identical to that of Flp.

[0014] In some embodiments, the amino acid sequence of the recombinase Cre or a variant thereof comprises any one of the sequences shown in SEQ ID NO: 3-5 or an amino acid sequence having more than 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) similarity.

[0015] In some implementations, the recombinase Cre variant includes Dre or VCre.

[0016] The sequences of SEQ ID NO: 3-5 are shown below: SEQ ID NO: 3: Cre / iCre SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCAYQDIRNFLLLLL RIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGVLLEDGVLL SEQ ID NO: 4: Dre MGASELIISGSSGGFLRNIGKEYQEAAENFMRFMNDQGAYAPNTLRDLRLVFHSWARWCHARQLAWFPISPEMAREYFLQLHDADLASTTIDKHYAMLNMLLSHCGLPPLSDKSVSLAMRRIRREAATEKGERTGQAIPLRWDDLKLLDVLLSRERVDLVDLVRLFRLRFRMRM SEISRIVGDLDQTGDTVTLHISHTKTITTTAAGLDKVLSRRTTAVLNDWLDVSGLREHPDAVLFPPIHRSNKARITTPLTPAMEKIFSDAWVLLNKRDATPNKGRYRTWTGHSARVGAAIDMAEKQVSMVEIMQEGTWKKPETLMRRGGVGANSVEIMQEGTWKKPETLMRRGGVGANSVAGKRSVKRSKVKKKKKKK SEQ ID NO: 5: VCre IENQLSLLGDFSGVRPDDVKTAIQAAQKKGINVAENEQFKAAFEHLLNEFKKREERYSPNTLRRLESAWTCFVDWCLANHRHSLPATPDTVEAFFIERAEELHRNTLSVYRWAISRVHRVAGCPDPCLDIYVEDRLKAIARKKVREGEAVKQASPFNEQHLLKLTSLWYRSDKLLLRRNLALLAVAYES MLRASELANIRVSDMELAGDGTAILTIPITKTNHSGEPDTCILSQDVVSSLLMDYTEAGKLDMSSDGFLFVGVSKHNTCIKPKKDKQTGEVLHKPITTKTVEGVFYSAWETLDLGRQGVKPFTAHSARVGAAQDLLKKGYNTLQIQQSGRWSSGAMVARYGRAILARDGAMAHSRVKTRSAPMQWGKDEKD In some embodiments, the nucleotide sequence expressing the recombinase FlpO or a variant thereof comprises the sequence shown in SEQ ID NO: 6 or other nucleotide sequences encoding a protein having 85% or more (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with any one of SEQ ID NO: 1-2. The sequence of SEQ ID NO: 6 is shown below: SEQ ID NO: 6: FlpO In some embodiments, the nucleotide sequence expressing the recombinase Cre or a variant thereof comprises the sequence shown in SEQ ID NO: 7 or other nucleotide sequences encoding a protein having 85% or more (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with any one of SEQ ID NO: 3-5. The SEQ ID NO: 7 sequence is shown below: SEQ ID NO: 7: Cre In some implementations, the dual recombinase is expressed via a CMV promoter.

[0017] In some embodiments, the adenovirus vector further includes the dual recombinase recognition site.

[0018] In some embodiments, the recognition site of the recombinase FlpO or a variant thereof is FRT.

[0019] In some embodiments, the recognition site of the recombinase Cre is loxP.

[0020] In some implementations, the recombinase Dre recognizes rox as its recognition site.

[0021] In some implementations, the recombinase VCr recognition site is VloxP.

[0022] In some embodiments, the FRT nucleotide sequence comprises a sequence as shown in SEQ ID NO: 8 or a nucleotide sequence having a sequence identity of 85% or more (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0023] In some embodiments, the loxP nucleotide sequence comprises a sequence as shown in SEQ ID NO: 9 or a nucleotide sequence having a sequence identity of 85% or more (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0024] In some embodiments, the rox nucleotide sequence comprises a nucleotide sequence as shown in SEQ ID NO: 10 or a nucleotide sequence having a sequence identity of 85% or more (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0025] In some embodiments, the VloxP nucleotide sequence comprises a sequence as shown in SEQ ID NO: 11 or a nucleotide sequence having a sequence identity of 85% or more (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0026] The sequences of SEQ ID NO: 8-11 are shown below: SEQ ID NO: 8: FRT GAAGTTCCTATACTTTCTAGAGAATAGGAACTTC SEQ ID NO: 9: loxP ATAACTTCGTATAGCATACATTATACGAAGTTAT SEQ ID NO: 10: rox TAACTTTAAATAATGCCAATTATTTAAAGTTA SEQ ID NO: 11: VloxP TCAATTTCTGAGAACTGTCATTCTCGGAAATTGA In some embodiments, the adenovirus vector further includes a linker arm, wherein the two recombinases are linked by the linker arm.

[0027] In some implementations, the connecting arm is 2A.

[0028] In some implementations, the connecting arm 2A includes P2A, T2A, F2A, or E2A.

[0029] In some embodiments, the P2A linker arm comprises an amino acid sequence as shown in SEQ ID NO: 12.

[0030] In some embodiments, the T2A linker arm comprises an amino acid sequence as shown in SEQ ID NO: 13.

[0031] In some embodiments, the E2A linker arm comprises an amino acid sequence as shown in SEQ ID NO: 14.

[0032] In some embodiments, the F2A linker arm comprises an amino acid sequence as shown in SEQ ID NO: 15.

[0033] The sequences of SEQ ID NO: 12-15 are shown below: SEQ ID NO: 12: P2A ATNFSLLKQAGDVEENPGP SEQ ID NO: 13: T2A EGRGSLLTCGDVEENPGP SEQ ID NO: 14: E2A QCTNYALLKLAGDVESNPGP SEQ ID NO: 15: F2A VKQTLNFDLLKLAGDVESNPGP In some embodiments, the mouse target cells include mouse embryo fibroblasts (MEF), alveolar epithelial cells (AT1 / 2), airway epithelial cells (ciliated cells, rod cells, basal cells, goblet cells), or cells derived from... Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze Other cell types in (Ai14) mice.

[0034] In some implementations, the adenovirus vector is Ad5.

[0035] In some implementations, the specific site is a mouse. Rosa26 Site.

[0036] In some implementations, the exogenous gene has no promoter.

[0037] In some implementations, the foreign gene is integrated into mice. Rosa26 Expression driven by promoters with site embedding.

[0038] In some implementations, the embedded promoter is a CAG promoter.

[0039] This approach ensures biosafety; if an operator ingests exogenous genes due to improper operation or other reasons, these genes will neither be expressed in the human body nor integrated into the genome.

[0040] In some embodiments, the exogenous gene is a reporter gene or a target gene, or a combination thereof. When both a reporter gene and a target gene are included, the target gene and the reporter gene may be connected by a linker arm.

[0041] In some implementations, the reporter gene is selected from one of EGFP, ZsGreen, or mNeonGreen.

[0042] In some embodiments, the target gene is selected from candidate or known gain-of-function variant genes in cancer, such as... EGFR , KRAS , NFE2L2 , CCDC Genes and their mutants or amplification events.

[0043] On the other hand, the present invention provides a method for preparing the above-mentioned adenovirus vector, wherein the method comprises the following steps: (1) Construct shuttle plasmids; (2) The shuttle plasmid was transformed into strain BJ5183 to produce a recombinant plasmid; (3) Transfect the recombinant plasmid into cells and package the linearized adenovirus vector.

[0044] In some embodiments, the shuttle plasmid comprises a foreign gene, a double recombinase FlpO or a variant thereof and Cre or a variant thereof, a double recombinase recognition site and a linker arm.

[0045] In some embodiments, the recombinant plasmid comprises a foreign gene, a double recombinase FlpO or a variant thereof and Cre or a variant thereof, a double recombinase recognition site and a linker arm.

[0046] In some embodiments, the cells are HEK-293A cells.

[0047] In some preferred embodiments, the shuttle plasmid comprises a foreign gene, FlpO, Cre, FRT, loxP, and 2A.

[0048] In some preferred embodiments, the recombinant plasmid contains a foreign gene, FlpO, Cre, FRT, loxP, and 2A.

[0049] In some implementations, the recombinant plasmid further includes a DNA identifier.

[0050] On the other hand, the present invention provides the use of the above-described adenovirus vector or the adenovirus vector prepared by the above method in the site-specific integration of exogenous genes.

[0051] In some embodiments, the integration occurs in mouse MEF cells, alveolar epithelial cells (AT1 / 2), airway epithelial cells (ciliated cells, rod cells, basal cells, goblet cells), or originates from... Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze Other cell types in (Ai14) mice.

[0052] In another aspect, the present invention provides the use of the above-described adenovirus vector or the adenovirus vector prepared by the above method in the preparation of a mouse exogenous gene expression model based on adenovirus vector.

[0053] In some embodiments, the use includes the step of delivering the adenovirus vector into a mouse.

[0054] In some implementations, the adenovirus vector is delivered into mice via endotracheal intubation.

[0055] In some implementations, the mouse strain is Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze (Ai14).

[0056] In some preferred embodiments, the Ad5-EGFP adenovirus vector is intubated into the endotracheal cannula. Gt(ROSA) 26Sor tm14(CAG-tdTomato)Hze (Ai14) in mice.

[0057] In some preferred embodiments, the Ad5-ZsGreen adenovirus vector is intubated into the endotracheal tube. Gt(ROSA) 26Sor tm14(CAG-tdTomato)Hze (Ai14) in mice.

[0058] In some implementations, the expression plasmids of multiple exogenous genes are introduced into the same plate cell or the same mouse to generate cells that overexpress multiple exogenous genes, which can significantly increase the throughput of gene research.

[0059] In some implementations, the amount of the foreign gene integrated into the cell is controlled by controlling the number of alleles in each cell.

[0060] In some implementations, each cell of an Ai14 / + heterozygous mouse integrates a single gene.

[0061] In some implementations, each cell of an Ai14 / Ai14 homozygous mouse integrates a single or two genes.

[0062] In some implementations, the model is a disease mouse model that can stably express exogenous genes.

[0063] In some implementations, the disease mouse model contains one or two integrated exogenous genes.

[0064] In some implementations, the disease mouse model is a tumor mouse model.

[0065] In some implementations, the tumor mouse model is used for oncology research, gene function research, research on the role of gene mutation, research on the role of gene overexpression, or research on drug response to specific genotype tumors.

[0066] In another aspect, the present invention provides the use of the above-described adenovirus vector or the adenovirus vector prepared by the above method in tumor research or tumor drug trials.

[0067] In some implementations, the adenovirus vector is used for oncology research, gene function research, research on the role of gene mutation or amplification, research on the role of gene overexpression, or research on drug response to specific genotype tumors.

[0068] In another aspect, the present invention provides the use of the above-mentioned adenovirus vector, disease mouse model, or adenovirus vector or disease mouse model prepared by the above method in the preparation of tumor drugs.

[0069] The tumors discussed in this article include both benign and malignant tumors, including tumors of the brain and central nervous system (such as astrocytoma, glioma, ependymoma, neuroblastoma, medulloblastoma, meningioma, pituitary adenoma, craniopharyngioma, and retinoblastoma), head and neck cancers (such as nasopharyngeal carcinoma, oral cancer, oropharyngeal cancer, laryngeal cancer, and thyroid cancer), lung cancer (including non-small cell lung cancer and small cell lung cancer), pleural tumors (such as malignant mesothelioma), mediastinal tumors (such as thymoma and thymic carcinoma), breast cancer, upper gastrointestinal tumors (such as esophageal cancer and gastric cancer), hepatobiliary and pancreatic tumors (such as liver cancer, intrahepatic bile duct cancer, gallbladder cancer, and pancreatic cancer), and intestinal tumors (such as small bowel cancer, colorectal cancer, and appendix cancer). Cancers include anal canal cancer, gastrointestinal stromal tumors, reproductive system tumors (such as prostate cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer), urinary system tumors (such as kidney cancer, bladder cancer, ureteral cancer), leukemia (such as acute lymphoblastic leukemia, chronic myeloid leukemia), lymphoma (such as Hodgkin lymphoma, non-Hodgkin lymphoma), plasma cell tumors (such as multiple myeloma), bone tumors (such as osteosarcoma, Ewing sarcoma), soft tissue sarcomas (such as liposarcoma, rhabdomyosarcoma), skin cancer (such as melanoma, basal cell carcinoma, squamous cell carcinoma), endocrine system tumors (such as adrenocortical carcinoma, pheochromocytoma), and cancers and carcinoid tumors of unknown primary origin.

[0070] The tumors described in this article include both benign and malignant tumors, including acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, anal cancer, appendiceal cancer, astrocytoma, atypical teratoma / rhabdoid tumor, basal cell carcinoma, bladder cancer, brainstem glioma, brain tumors (including brainstem glioma, atypical teratoma / rhabdoid tumor of the central nervous system, embryonal tumors of the central nervous system, astrocytoma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medullary epithelioma, and intermediate... Differentiated pineal parenchymal tumors, supratentorial primitive neuroectodermal tumors and pinealoblastomas), breast cancer, bronchial tumors, Burkitt lymphoma, cancers of unknown primary location, carcinoid tumors, malignant tumors of unknown primary location, atypical teratomas / rhabdoid tumors of the central nervous system, embryonal tumors of the central nervous system, cervical cancer, childhood cancers, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, endocrine islet cell tumors, endometrial cancer. Ependymoma, ependymoma, esophageal cancer, nasal glioma, Ewing sarcoma, extracranial germ cell tumor, gonadal germ cell tumor, extrahepatic bile duct carcinoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastoma, glioma, hairy cell leukemia, head and neck cancer, cardiac cancer, Hodgkin's lymphoma, pharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer Liver cancer, lung cancer, malignant fibrous histiocytoma, bone cancer, medulloblastoma, medullary epithelioma, melanoma, Merkel cell carcinoma, Merkel cell skin cancer, mesothelioma, occult primary metastatic squamous neck cancer, multiple endocrine adenoma syndrome, multiple myeloma, multiple myeloma / plasma celloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic tumors, nasal cavity cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, oral cancer, oral cavity cancerCancer), oropharyngeal cancer, osteosarcoma, other brain and spinal cord tumors, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumors, low-grade potential ovarian tumors, pancreatic cancer, papillomatosis, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, pharyngeal cancer, moderately differentiated pineal parenchymal tumors, pineal cell tumors, pituitary adenomas, plasmacytoma / multiple myeloma, pleural mastoblastoma, primary central nervous system (CNS) lymphoma, primary hepatocellular carcinoma, prostate cancer, rectal cancer, kidney cancer, kidney Cellular carcinoma, respiratory tract cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sézary syndrome, small cell lung cancer, small bowel cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, T-cell lymphoma, testicular cancer, laryngeal cancer, thymic carcinoma, thymoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumors, ureteral cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, perineal cancer, Waldenström macroglobulinemia, or Wilms' tumor; preferably lung cancer.

[0071] In another aspect, the present invention provides a disease mouse model prepared according to the above method.

[0072] In some implementations, the disease mouse model is a disease mouse model that can stably express exogenous genes.

[0073] Advantages of this invention Existing technologies either deliver vectors such as plasmids and viruses through invasive injection / electroconvulsion or utilize lentiviruses. This invention delivers exogenous genes via adenovirus vector through endotracheal intubation, utilizing dual recombinases for site-specific integration into the mouse Rosa26 site. This method is highly efficient, with controllable integration sites and copy numbers, good safety profile, and can efficiently model various gain-of-function variants.

[0074] The exogenous genes delivered in this invention have no promoter; they are expressed only after integration into the Rosa26 site of the Ai14 mouse strain, driven by the mouse Rosa26 enhancer and CAG promoter, ensuring biosafety. If an operator ingests the exogenous genes due to improper handling or other reasons, these genes will neither be expressed in the human body nor integrated into the genome. This design is safer than previous lentivirus-based technologies. Attached Figure Description

[0075] Figure 1The diagram shows the design of the shuttle plasmid used to construct the adenovirus plasmid in Example 1. PA: polyadenylation; BFP: blue fluorescent protein; ITR: terminal inverted repeat sequence, which is the initiation site of adenovirus replication; Pme I: restriction enzyme site; FRT site is the FlpO recombinase recognition site; loxP site is the Cre recombinase recognition site; the two recombinases and BFP are connected by a linker arm. The vector also contains packaging signal (ψ) sequences.

[0076] Figure 2 The diagram shows the plasmid structure used to prepare the adenovirus vector in Example 1. PA: polyadenylated acid; BFP: blue fluorescent protein; ITR: terminal inverted repeat sequence, the initiation site for adenovirus replication; Pac I: restriction enzyme site; FRT site: FlpO recombinase recognition site; loxP site: Cre recombinase recognition site; the two recombinases and BFP are connected by a linker arm.

[0077] Figure 3 Preparation and amplification of the adenovirus vector in Example 1. (A) Gel electrophoresis verification of pShuttle-ZsGreen shuttle plasmid and pAdeasy-ZsGreen recombinant plasmid. (B) Pac I restriction enzyme digestion results of the recombinant plasmid. (C) Results of transfection of the Pac I linearized recombinant plasmid into HEK293A. Blue fluorescence is from blue fluorescent protein (BFP), scale bar is 50 μm.

[0078] Figure 4 The diagram shows the principle of site-specific integration of exogenous genes in mice in Examples 1 and 2.

[0079] Figure 5 The image shows the site-specific integration of the ZsGreen gene into Ai14 homozygous MEF cells mediated by the adenovirus vector and dual recombinase system in Example 1. The scale bar is 50 μm.

[0080] Figure 6 The image shows the site-specific integration and expression of the ZsGreen gene in the lungs of Ai14 homozygous mice mediated by the adenovirus vector and dual recombinase system in Example 2. (A) Results of site-specific integration and expression of the reporter gene ZsGreen in frozen sections of mouse lungs, scale bar 10 μm. (B) Immunohistochemical results of mouse lung tissue; the brown cells indicated by the arrows represent the site-specific integration and expression of the reporter gene ZsGreen, scale bar 25 μm. (C) If the mice are replaced with C57Bl6 / J wild-type mice, the ZsGreen reporter gene cannot be integrated, and there is no green or red fluorescence, scale bar 1 cm.

[0081] Figure 7The image shows a mouse lung cancer model constructed using an adenovirus vector and a dual recombinase system mediated by ZsGreen gene integration in Example 3. (A) Mouse orthotopic lung cancer model constructed using the method described above. Red indicates tumors successfully delivered Cre, and green indicates tumors integrating the reporter gene ZsGreen. The scale bar is 1 cm. (B) Results of frozen sections of lung tissue. The scale bar is 50 μm. (C) Results of H&E staining of mouse lung tissue. The scale bars are 100 μm (panoramic view) and 25 μm (small image), respectively. (D) Immunohistochemical results of mouse lung tissue. The red arrow indicates the expression region of the exogenous gene. The scale bar is 25 μm. Detailed Implementation

[0082] For the purpose of clarity and concise description, features are described herein as part of a number of identical or separate embodiments. However, it will be understood that the scope of this disclosure may include embodiments having combinations of all or some of the described features. The technical solutions of the present invention will now be described clearly and completely. Obviously, based on the specific embodiments of the present invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention.

[0083] I. Definition Unless otherwise specified, the following terms used in the specification and claims shall have the following meanings: As used herein, the terms “comprising” or “including” mean “including, but not limited to”. This term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term “comprising” includes the more restrictive terms “consisting of” and “substantially consisting of”. In one embodiment, the term “comprising” as used throughout the application, particularly in the claims, may be replaced by the term “consisting of”.

[0084] As used herein, the terms “optional,” “any,” “arbitrary,” or “any one” mean that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation occurs or does not occur. As used herein, “an” and “a” refer to one or more grammatical objects.

[0085] As used in this article, “and / or” should be understood as meaning any one of the options or any combination of two or more of the options.

[0086] As used in this article, the term "adenovirus" refers to a non-enveloped, icosahedral, linear double-stranded DNA virus, approximately 35 kbp in size. Vectors based on human adenovirus type 5 (Ad5) are widely used. These vectors are characterized by the artificial deletion of the E1 and E3 genes, resulting in the adenovirus losing its replication ability and reducing its immunogenicity to some extent. This makes Ad5 a widely used, safe, and effective gene delivery vector.

[0087] As used herein, the term "gene" means a DNA sequence that is or includes a coding sequence (i.e., a DNA sequence encoding an RNA product, which can be further translated into a polypeptide and folded into a protein), optionally together with some or all of the regulatory sequences controlling the expression of the coding sequence. In some embodiments, a gene includes non-coding sequences, such as, but not limited to, introns. In some embodiments, a gene may include both coding (e.g., exons) and non-coding (e.g., introns) sequences. In some embodiments, a gene includes a regulatory sequence that serves as a promoter. The term "exogenous gene" refers to a gene delivered into the cells of a host organism. The expression of exogenous genes is typically achieved through genetic engineering techniques, such as gene cloning, gene transfer, and gene expression, to alter the genetic characteristics of the host organism or to generate new functions, and if integrated into open regions of chromatin, can be expressed long-term. In some embodiments, the exogenous gene is a tumor-related gene, such as KRAS, SOX2, etc.

[0088] As used herein, the term "vector" refers to a nucleic acid molecule or replication-defective adenovirus (including a protein capsid and a double-stranded DNA carrying the designed sequence) capable of propagating. In some embodiments, the vector directs the expression of the gene it carries. Such vectors are referred to herein as "expression vectors".

[0089] As used herein, the term "recombinase" refers to a protein that catalyzes recombination. The term "recombination" refers to the excision, inversion, integration, chromosomal exchange, or rearrangement of DNA in a target DNA sequence, such as the host genome. In some embodiments, the construction methods described herein use dual recombinases; in some embodiments, the dual recombinases are Cre and FlpO.

[0090] As used in this article, the term "cell" can refer to cells of human or non-human animal origin, such as mouse embryonic fibroblasts (MEF) of mouse origin and HEK-293A cells of human embryonic kidney cell origin.

[0091] As used herein, the term "identity" has a generally accepted meaning in the art, and the percentage of sequence similarity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule.

[0092] As used herein, the term "transfection" refers to various techniques for introducing exogenous nucleic acid molecules into prokaryotic or eukaryotic host cells. In some embodiments, a recombinant plasmid containing said exogenous nucleic acid molecule is transfected into 293A cells to package a linearized adenovirus vector. The term "transduction" is a specific type of transfection in which genetic material is transferred from one source to another, such as via a virus (e.g., adenovirus or lentivirus) or a transduced bacteriophage. When the term is used to refer to the introduction of nucleic acid molecules into microbial cells such as bacteria and yeast, the term "transformation" may be used interchangeably with "transfection". In some embodiments, said exogenous nucleic acid molecules are transduced into MEF cells via an adenovirus vector. The term "transformation" is used herein to describe genetic changes resulting from the acquisition of exogenous nucleic acids by microorganisms and is substantially synonymous with "transfection". Transfection techniques include, but are not limited to, transformation, transduction, particle bombardment, diffusion, active transport, ultrasonic bath, electroporation, microinjection, lipid transfection, adsorption, infection, protoplasmic fusion, calcium phosphate precipitation, DEAE-glucan transfection, etc.

[0093] As used herein, the term “expression” refers, individually and / or cumulatively, to one or more biological processes that result in the production of coding agents such as proteins from nucleic acid sequences. Expression specifically includes either or both of transcription and translation.

[0094] II. Examples The present invention will be described in detail below through specific embodiments. It should be understood that the following embodiments are for explanation and illustration only and do not limit the scope of the present invention in any way.

[0095] In the following embodiments, unless otherwise specified, all biochemical reagents are conventional reagents in the art, which can be prepared according to conventional methods in the art or obtained commercially, and the specification is laboratory grade.

[0096] Table 1 Example 1: Construction and validation of adenovirus vector in cell models Adenovirus vectors are commonly used expression vectors in the fields of genetics and cell biology. Their titers are often much higher than those of lentiviral vectors, resulting in high transduction and expression efficiency and a large carrying capacity (~8 kb). They generally do not spontaneously integrate into the genome and are often used for transient expression. They are widely used in modeling various lung cancer subtypes, such as small cell lung cancer.

[0097] First, construct the shuttle plasmid pShuttle-Gene, which encodes the exogenous gene and the double recombinase. Figure 1 The shuttle plasmid was then transformed into strain BJ5183 to construct the pAdeasy-Gene. The design of the pAdeasy-Gene is as follows: Figure 2As shown. Taking the reporter gene ZsGreen as an example, after constructing pAdeasy-ZsGreen, agarose gel electrophoresis was used to identify that the molecular weight of the pAdeasy-ZsGreen recombinant plasmid was larger than that of the shuttle plasmid pShuttle-ZsGreen (…). Figure 3 A). The pAdeasy-ZsGreen plasmid was extracted and digested with PacI to obtain a 4500 bp fragment, such as... Figure 3 As shown in Figure B, the correct band length was verified by gel electrophoresis, proving successful plasmid recombination. Subsequent first-generation sequencing confirmed the sequence was correct. The linearized pAdeasy-ZsGreen recombinant plasmid was then transfected into HEK293A cells to prepare the Ad5-ZsGreen adenovirus vector. HEK293A cells are a cell line specifically designed for packaging and amplifying adenovirus. pAdeasy-ZsGreen expresses BFP blue fluorescence intracellularly to identify cells containing adenovirus, as shown in Figure B. Figure 3 As shown in Figure C, blue fluorescence can be observed under a fluorescence microscope 14 days after transfection, and obvious plaques can be observed in bright field, indicating that the adenovirus vector was successfully prepared.

[0098] The adenovirus vector technology route of this invention is as follows: Figure 4 As shown, the adenovirus vector carries a reporter gene or a target gene, and these genes have no promoter preceding them. This means that after the adenovirus vector enters the cell, the gene will not be expressed in the cell before integration into the genome, ensuring biosafety when using this vector to study oncogenes. The adenovirus vector also carries two recombinases driven by the CMV promoter. When the two recombinases are expressed, the reporter gene (e.g., ZsGreen) or the target gene (e.g., gain-of-function variant genes such as oncogenes) can be site-directedly integrated into Ai14 mice. Rosa26 site, in Rosa26 Driven by the enhancer and CAG promoter at the site, cells can stably and long-term express this gene. If site-directed integration does not occur, Rosa26 Only Cre-mediated deletion occurs at the site, thereby activating the expression of the tdTomato gene and producing red fluorescent protein.

[0099] Ai14 / Ai14 MEF cells were transduced using the Ad5-ZsGreen adenovirus vector. The control group used an Ad5 adenovirus vector that did not contain the exogenous ZsGreen gene. After 7 days, cells in the Ad5-ZsGreen group expressed ZsGreen, while cells in the control group showed no ZsGreen expression, only red fluorescence of tdTomato. This indicates that transduction using the Ad5-ZsGreen adenovirus vector can successfully integrate ZsGreen into the MEF cell genome. Figure 5 ).

[0100] Example 2: Construction of a mouse lung exogenous gene-targeted integration model Example 1 verified that the present invention can integrate the ZsGreen reporter gene into MEF cell lines. Example 2 further constructed a mouse lung exogenous gene site-specific integration model.

[0101] 60-100 μL of a 10% concentration was administered via endotracheal intubation. 6 -10 12 Adenovirus vector at pfu / mL was delivered to the lungs of Ai14 mice, along with a control group adenovirus of the same concentration (containing only recombinase and no exogenous gene). After 3 weeks, the lung tissue of the mice was observed. If the exogenous gene underwent site-directed integration, the cells would stably and long-term express the gene. If site-directed integration did not occur, the cells would only produce red fluorescent protein.

[0102] Taking the ZsGreen reporter gene as an example, Ad5-ZsGreen was delivered to the lung tissue of Ai14 / Ai14 genetically engineered mice via endotracheal intubation. Three weeks later, frozen sections of the mouse lung tissue showed green fluorescence expression in alveolar epithelial cells, while the control group only showed red fluorescence. Figure 6 A); Immunohistochemical results showed that ZsGreen protein was expressed in mouse lung tissue ( Figure 6 B), the control group did not express ZsGreen protein. These results demonstrate that the present invention can achieve the integration and long-term expression of exogenous genes in mouse lung tissue. When Ad5-ZsGreen was delivered to C57Bl6 / J wild-type mice via endotracheal intubation, no green fluorescence was observed because the mice did not carry the Ai14 gene, the adenovirus did not integrate the reporter gene, and no green fluorescence was observed, indicating that the system possesses Ai14 site specificity and biosafety. Figure 6 C).

[0103] Example 3: Construction of a mouse lung cancer model with site-specific integration of reporter genes Example 2 verifies that the present invention can achieve site-specific integration of exogenous genes in the mouse lungs. Example 3 utilizes the present invention to construct a mouse lung cancer model with site-specific integration of exogenous genes (reporter genes).

[0104] Adenovirus vector (Ad5-ZsGreen) was delivered into genetically engineered mice via endotracheal intubation. Kras LSL-G12D / + ; Rosa26 LSL-tdTomato / + In the lungs, this genotype of mouse can delete the LSL component (LSL is a transcription termination sequence with Cre binding sites at both ends) after Cre activation, thus activating... Kras G12D / +The adenovirus vector contains a green fluorescent protein (Green) gene (ZsGreen) and two different recombinase genes (Cre and FlpO). These two recombinases help the Green Fluorescent Protein (Green) gene integrate into a specific site in the mouse genome (Rosa26), and Green Fluorescent Protein (Green) indicates whether the ZsGreen gene integration is complete. All three genes are transduced into mouse lung cells. Both recombinases work simultaneously, with the Green Fluorescent Protein (Green) gene integrating into the mouse lung cells and emitting green light. The Cre recombinase mediates the deletion of two LSL structures, activating the Kras mutant gene. The green fluorescent mouse lung cells proliferate abnormally, evolving into green fluorescent tumors. A polyA structure is added to the tail of the ZsGreen gene to inhibit tdTomato expression, thus preventing red fluorescence. If the adenovirus only carries the Cre recombinase (Ad5-Cre), the Cre recombinase mediates the deletion of two LSL structures, and the mouse lung cells, while activating the Kras mutant gene, express the red fluorescent protein tdTomato, resulting in red fluorescent tumors.

[0105] Stereoscopic results showed that the mouse lungs produced tumors with green fluorescence and red fluorescence, while the control group (Ad5-Cre) only showed red fluorescent protein. Figure 7 A) indicates that the dual recombinase can integrate ZsGreen into the target site. Rosa26 The site was identified, while tumors with only Cre recombinase could only produce red fluorescence. This strain was expected to produce a single copy of the foreign gene integration; although some tumors showed simultaneous red and green fluorescence, frozen sections of the lungs showed that red and green fluorescence existed in different cells within the tumor. Figure 7 (B) This result indicates that site-specific integration of the exogenous gene prevented the expression of red fluorescent tdTomato. The control group only showed cells with red fluorescence. H&E staining results of mouse lung tissue showed that tumor cells were loosely arranged, forming irregular glandular structures; the cell nuclei were of uneven size and irregular shape, and some cells had large nucleoli, indicating that the cells had high proliferative activity (pathological characteristics). Figure 7 C), Immunohistochemistry showed that the tumor expressed ZsGreen protein, mainly distributed in the cytoplasm and nucleus, exhibiting a diffuse appearance. Figure 7 (D) No ZsGreen protein expression was detected in the control group, and immunohistochemical results further confirmed that ZsGreen was expressed in the tumors of the experimental group. These results demonstrate that the present invention can construct a mouse lung tumor site-specific integration model.

[0106] The exogenous gene delivered in this invention can be a gain-of-function variant gene such as an oncogene. If this gene is specifically integrated into mouse somatic cells of a particular genotype, the oncogene can promote the transformation of lung epithelial cells into cancer cells, leading to tumor formation and thus rapidly constructing mouse lung cancer models of different genotypes. This method is also applicable to the construction of other tumor models.

[0107] This invention can also significantly increase the throughput of gene research by introducing expression plasmids of multiple genes into the same plate cell or the same mouse somatic cell, generating mouse models expressing multiple exogenous genes, such as mouse lung cancer models. This invention can combine DNA barcoding for phenotypic quantification of mouse lung tumors, increasing the throughput of gene function research and improving the efficiency of constructing mouse models with gain-of-function mutations. Furthermore, the copy number of the integrated gene is controllable, depending on the Ai14 mouse chromosome. Rosa26 The knock-in tdTomato gene is either heterozygous or homozygous. The mouse chromosome set is diploid. Rosa26 There are two loci. By controlling the number of Ai14 alleles in each cell, the number of genes integrated into the cell can be controlled. Heterozygotes integrate a single gene, while homozygotes integrate up to two genes, thus allowing for the specific study of the function of single genes or gene combinations. This invention can be applied to the quantitative study of drug responses in various genotypes of tumors.

[0108] Because it uses a transiently expressed adenovirus vector, it does not spontaneously and randomly integrate into the genome, making this invention safer than traditional lentiviral techniques. Furthermore, since the delivered oncogenes and other gain-of-function variants lack a promoter, they only integrate into mice with specific genotypes. Rosa26 Expression can only occur after the site is identified, further ensuring the safety of the experimenters.

[0109] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention are covered within the scope of the present invention.

Claims

1. An adenovirus vector, characterized in that, The vector contains a foreign gene and a gene expressing a double recombinase, and can use the double recombinase to integrate the foreign gene into a specific chromatin site of mouse target cells.

2. The adenovirus vector according to claim 1, wherein, The dual recombinase is recombinase FlpO or a variant thereof and recombinase Cre or a variant thereof; Preferably, the recombinase FlpO variant includes FlpE or Flp; Preferably, the recombinase Cre variant includes Dre or VCre; Preferably, the amino acid sequence of the recombinase FlpO or its variants includes any one of the sequences shown in SEQ ID NO: 1-2 or an amino acid sequence having more than 85% identity with it; Preferably, the amino acid sequence of the recombinase Cre or its variants includes any one of the sequences shown in SEQ ID NO: 3-5 or an amino acid sequence having more than 85% identity with it; Preferably, the nucleotide sequence of the gene expressing the recombinase FlpO or a variant thereof includes the sequence shown in SEQ ID NO: 6, or other nucleotide sequences encoding a protein having more than 85% sequence identity with any one of SEQ ID NO: 1-2; Preferably, the nucleotide sequence of the gene expressing the recombinase Cre or a variant thereof includes the sequence shown in SEQ ID NO: 7, or other nucleotide sequences encoding a protein having more than 85% sequence identity with any one of SEQ ID NO: 3-5; Preferably, the dual recombinase is expressed via a CMV promoter.

3. The adenovirus vector according to claim 1 or 2, wherein, The adenovirus vector further includes the dual recombinase recognition site; Preferably, the recognition site of the recombinase FlpO or its variant is FRT; Preferably, the recognition site of the recombinase Cre is 10xP; Preferably, the recombinase Dre recognition site is rox; Preferably, the recombinase VCr recognition site is VloxP; Preferably, the FRT nucleotide sequence comprises the sequence shown in SEQ ID NO: 8 or a nucleotide sequence having more than 85% sequence identity with it; Preferably, the loxP nucleotide sequence comprises the sequence shown in SEQ ID NO: 9 or a nucleotide sequence having more than 85% sequence identity with it; Preferably, the rox nucleotide sequence comprises the sequence shown in SEQ ID NO: 10 or a nucleotide sequence having more than 85% sequence identity with it; Preferably, the VloxP nucleotide sequence comprises the sequence shown in SEQ ID NO: 11 or a nucleotide sequence having more than 85% sequence identity with it.

4. The adenovirus vector according to any one of claims 1-3, wherein, The exogenous gene has no promoter; Preferably, the specific site is a mouse. Rosa26 site; Preferably, the foreign gene is integrated into mice. Rosa26 Expression driven by promoters with site embedding; Preferably, the promoter is a CAG promoter; Preferably, the exogenous gene is a reporter gene or a target gene, or a combination thereof; Preferably, the reporter gene is selected from one of EGFP, ZsGreen, or mNeonGreen; Preferably, the target gene is selected from gain-of-function variant genes in cancer; More preferably, the target gene comprises EGFR, KRAS, CCND, or SOX2 Genes and their mutants or amplification events.

5. The use of the adenovirus vector according to any one of claims 1-4 in site-specific integration of exogenous genes; Preferably, the integration occurs in mouse embryonic fibroblasts, alveolar epithelial cells, airway epithelial cells, or originates from... Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze Other cell types in (Ai14) mice.

6. Use of the adenovirus vector according to any one of claims 1-4 in the preparation of a mouse exogenous gene expression model based on an adenovirus vector.

7. The use according to claim 6, wherein, Adenovirus vectors containing multiple exogenous genes were introduced into the same plate cells or the same mouse to generate cells that overexpress multiple exogenous genes.

8. The use according to claim 6 or 7, wherein, The number of foreign genes integrated into the cell can be controlled by controlling the number of alleles in each cell. Preferably, each cell of the Ai14 / + heterozygous mouse integrates a single gene; Preferably, each cell of the Ai14 / Ai14 homozygous mouse integrates a single gene or two genes.

9. The use according to any one of claims 6-8, wherein, The model is a disease mouse model that can stably express exogenous genes; Preferably, the disease mouse model contains one or two integrated exogenous genes; Preferably, the disease mouse model is a tumor mouse model; Preferably, the tumor mouse model is used for oncology research, gene function research, research on the role of gene mutation, research on the role of gene overexpression, or research on drug response to specific genotype tumors; More preferably, the tumor is lung cancer.

10. Use of the adenovirus vector according to any one of claims 1-4 in tumor research or tumor drug trials; Preferably, the adenovirus vector is used for oncology research, gene function research, research on the role of gene mutation or amplification, research on the role of gene overexpression, or research on drug response of specific genotype tumors. Preferably, the tumor is lung cancer.

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