CN116716349A - Construction method and application of DLL4 humanized mouse model - Google Patents

Construction method and application of DLL4 humanized mouse model Download PDF

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CN116716349A
CN116716349A CN202310958263.2A CN202310958263A CN116716349A CN 116716349 A CN116716349 A CN 116716349A CN 202310958263 A CN202310958263 A CN 202310958263A CN 116716349 A CN116716349 A CN 116716349A
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邢俊
汪慧怡
琚存祥
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Jiangsu Jicui Yaokang Biotechnology Co ltd
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Abstract

The invention relates to a construction method of a DLL4 humanized mouse model, which comprises the following steps: (1) constructing a targeting vector for expressing the humanized DLL 4; (2) Designing and obtaining sgrnas for the Exon1 to Exon9 portions of the mouse Dll4 gene; (3) Co-injecting or co-electrotransferring the targeting vector, sgRNA and Cas9 protein into cytoplasm or nucleus of fertilized egg of mouse, transplanting fertilized egg into pseudopregnant mouse, and carrying out genotype identification to pseudopregnant mouse, and screening positive F0 mouse successfully inserted into correct humanized fragment; (4) F0 mice and background mice are bred to obtain F1 mice, and DLL4 humanized mouse models are screened. The DLL4 humanized mice constructed by the invention have application value in the fields of oncology, immunology and the like.

Description

Construction method and application of DLL4 humanized mouse model
Technical Field
The invention relates to the field of animal genetic engineering, in particular to a construction method and application of a DLL4 humanized mouse model.
Background
The concept of tumor immunotherapy has been increasingly known in recent years, but the development of novel immunotherapeutic drugs still faces many challenges. Since antibodies to general specific human proteins are unable to recognize mouse endogenous proteins, normal wild-type mice are unable to serve as in vivo models for testing these drugs. Due to species-specific limitations, anti-mouse antibody formulations do not reflect the actual efficacy of the same biological agents in humans. These problems have hampered preclinical evaluation of immunotherapy and limited the progress in the development of new tumor immune drugs. Accordingly, researchers have begun to focus on the development of humanized mouse models.
Humanized mice are mice carrying functional human genes, cells, tissues and/or organs, immune systems or microorganisms, which are used in biomedical research and development of clinical treatment protocols. Some humanized mice carry human cells, and some have certain consistent genetic and physiological properties with humans. Because the mouse genome is highly similar to the human genome and is easier to operate and change, the mouse can conveniently simulate the biological characteristics of human beings and human diseases, thereby helping research and development personnel to break the pathogenic principle and the molecular mechanism thereof and guiding the development of medicaments.
Delta-like protein 4 (DLL 4) is a type I transmembrane protein whose extracellular domain comprises a Delta and Serrate (DSL) ligand domain and eight tandem epidermal growth factor-like repeats. Upon binding to its ligands Notch 1 and Notch 4, the extracellular and cytoplasmic domains are cleaved and the cytoplasmic domains are transferred into the nucleus. DLL4 is expressed at both physiological and pathological sites of angiogenesis. Mice carrying the lacz reporter under the control of the Dll4 promoter showed a significant increase in Dll4 expression in the tumor vasculature. In human tumors, DLL4 is expressed in the vasculature of clear cell renal cell carcinoma, as well as in superficial and invasive bladder cancers. VEGF and DLL4 expression are closely related in cultured endothelial cells, preclinical tumor models, and human tumor samples. From these observations, we easily speculate that the DLL4/Notch pathway may be involved in tumor angiogenesis.
DLL4 is not only critical in vascular development and homeostasis, but also in the formation of tumor functional vascular networks. Blocking Dll 4-mediated Notch signaling can significantly increase non-productive angiogenesis but significantly inhibit tumor growth in preclinical mouse models. Thus, DLL4 has become an attractive target for cancer treatment. anti-DLL 4 antibodies have recently entered the clinical trial phase. However, the potential toxic effects of anti-DLL 4 are not clear. It has been reported in the literature that chronic DLL4 blocks abnormal activation of endothelial cells, causes multiple organ pathological changes, and induces vascular tumors. These findings need to be confirmed in studies using different tumor animals, but pose significant safety issues for the use of anti-DLL 4 drugs and deserve monitoring of these effects in clinical trials targeting DLL 4.
Humanized mice can mimic human diseases and are therefore commonly used in the study of infectious, degenerative and cancer diseases. Recent models also reflect hematopoietic, natural immune, neurobiological and molecular pathways affecting disease pathology biology. A series of immunodeficient mouse strains allow for long-lived human progenitor cell transplantation. The presence of innate and adaptive immunity enables the reconstruction of high levels of human lymph, cells being susceptible to a wide range of microbial infections. These mice also contribute to the study of human pathobiology, natural disease processes and the therapeutic effects of a wide range of human diseases. Therefore, the gene coding the human DLL4 is knocked into the homologous C57BL/6JGpt mouse locus by utilizing a gene editing means, a mouse model for expressing the human DLL4 cytokine can be constructed, and the mouse model has application value in researching immunotherapy, inflammation, autoimmune diseases and related drug screening.
At present, a construction method of a DLL4 humanized mouse model and related literature reports of the DLL4 humanized mouse model in the aspect of target drug application are not seen.
Disclosure of Invention
In view of the problems existing at present, a first aspect of the present invention provides a method for constructing a DLL4 humanized mouse model, the method comprising the steps of:
(1) Constructing a targeting vector for expressing the humanized DLL4, and inserting the humanized DLL4 gene;
(2) Designing sgrnas for the Exon1 to Exon9 parts of the mouse Dll4 gene, and obtaining the sgrnas by using an in vitro transcription technology;
(3) Co-injecting or co-electrotransferring the targeting vector constructed in the step (1), the sgRNA obtained in the step (2) and the Cas9 protein into cytoplasm or nucleus of fertilized ovum of the mouse, transplanting the fertilized ovum into pseudopregnant mouse, carrying out genotype identification on pseudopregnant mouse, and screening positive F0 mouse successfully inserted into correct human fragment;
(4) F0 mice and background mice are bred to obtain F1 mice, the tail of the F1 mice is subjected to gene identification, and a DLL4 humanized mouse model is screened.
Preferably, the step (1) includes the following steps: according to the structure and function of the human DLL4 gene, the signal peptide and extracellular region of the human DLL4 gene are selected to replace the signal peptide and extracellular region sequence of the murine Dll4 gene, the amino acid sequence of the selected human DLL4 gene is shown as SEQ ID No.1, and the amino acid sequence of the replaced murine Dll4 gene is shown as SEQ ID No. 2.
Preferably, the step (1) includes the following steps: the Exon1 to Exon9 parts of the human DLL4 gene are selected to replace the Exon1 to Exon9 parts of the mouse Dll4 gene, and the sequence of the selected human DLL4 gene is shown as SEQ ID No. 3.
Preferably, the sequence of the targeting vector successfully constructed in the step (1) is shown as SEQ ID No. 4.
Preferably, the sgRNA in step (2) has the gene sequence of (a) SEQ ID NO.5 and SEQ ID NO.7, or (b) SEQ ID NO.6 and SEQ ID NO.8.
More preferably, the sgRNA in the step (2) has the gene sequences of SEQ ID NO.6 and SEQ ID NO.8.
Preferably, the mice provided with fertilized eggs in the step (3) and the pseudopregnant mice are B6 mice.
Preferably, the 5 'end identification primer used for F0 mouse genotype identification in the step (3) is shown as SEQ ID NO.9 and SEQ ID NO.10, and the 3' end identification primer is shown as SEQ ID NO.11 and SEQ ID NO. 12.
Preferably, the PCR reaction system used for genotyping the F0 mice in the step (3) is as follows:
。
preferably, the PCR reaction conditions used for genotyping the F0 mice in the step (3) are as follows:
。
the second aspect of the present invention provides the use of the mice obtained by the above construction method for studying DLL4 gene-related functions and mechanisms of action.
Preferably, the use is for non-diagnostic and non-therapeutic purposes.
In a third aspect, the present invention provides the use of the mice obtained by the above construction method for screening drugs for treating diseases associated with the DLL4 gene.
Preferably, the use is for non-diagnostic and non-therapeutic purposes.
The invention has the beneficial effects that:
according to the comparison of human DLL4 protein functional domains and human murine homology, the CRISPR/Cas9 technology is used for replacing the signal peptide and extracellular domain of the murine Dll4 gene with the corresponding regions of the human DLL4 gene on mice with C57BL/6JGpt (B6) background. The chimeric DLL4 gene sequence will be expressed under the direction of endogenous regulatory mechanisms. The model has application value in the fields of oncology, immunology and the like.
Drawings
FIG. 1 is a schematic of a DLL4 humanized mouse model;
FIG. 2 is an electrophoretogram of the identification of the 5 'and 3' end genes of the DLL4-KI-target F0 mice;
FIG. 3 is an electrophoretogram of the identification of the 5 'and 3' end genes of the DLL4-KI-target F1 mice;
FIG. 4 shows the results of the detection of mRNA expression in B6-hDLL4 homozygous mice;
FIG. 5 shows the results of detection of the expression of the B6-hDLL4 homozygous mouse DLL4 protein.
Detailed Description
The present invention is further illustrated by way of examples, but the present invention is not limited to the following examples.
Test example 1, establishment of DLL4 humanized mouse model
The invention uses CRISPR/Cas9 technology to replace signal peptide and extracellular domain of murine Dll4 gene with corresponding region of human DLL4 gene on C57BL/6JGpt (B6) background mice (specific strategy is shown in figure 1), chimeric DLL4 gene sequence is expressed under the guidance of endogenous regulation mechanism, thus constructing mouse model capable of expressing human DLL4, and the specific method is as follows:
1. determination of human fragment substitution region and inserted human sequence
According to the structure and function of the human DLL4 gene, the signal peptide of the human DLL4 gene and the signal peptide and extracellular region sequence of the extracellular region of the replaced murine Dll4 gene are selected, the amino acid sequence of the selected human DLL4 gene is shown as SEQ ID No.1 (Aa: 1-529), and the amino acid sequence of the replaced murine Dll4 gene is shown as SEQ ID No.2 (Aa: 1-529).
MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCYTDLSTDTFVCNCPYGFVGSRCEFPVGLPPSFPW （SEQ ID No.1）
MTPASRSACRWALLLLAVLWPQQRAAGSGIFQLRLQEFVNQRGMLANGQSCEPGCRTFFRICLKHFQATFSEGPCTFGNVSTPVLGTNSFVVRDKNSGSGRNPLQLPFNFTWPGTFSLNIQAWHTPGDDLRPETSPGNSLISQIIIQGSLAVGKIWRTDEQNDTLTRLSYSYRVICSDNYYGESCSRLCKKRDDHFGHYECQPDGSLSCLPGWTGKYCDQPICLSGCHEQNGYCSKPDECICRPGWQGRLCNECIPHNGCRHGTCSIPWQCACDEGWGGLFCDQDLNYCTHHSPCKNGSTCSNSGPKGYTCTCLPGYTGEHCELGLSKCASNPCRNGGSCKDQENSYHCLCPPGYYGQHCEHSTLTCADSPCFNGGSCRERNQGSSYACECPPNFTGSNCEKKVDRCTSNPCANGGQCQNRGPSRTCRCRPGFTGTHCELHISDCARSPCAHGGTCHDLENGPVCTCPAGFSGRRCEVRITHDACASGPCFNGATCYTGLSPNNFVCNCPYGFVGSRCEFPVGLPPSFPWVA （SEQ ID No.2）
2. Injection to obtain positive mice
The Exon1 to Exon9 portions of the humanized DLL4 gene were replaced with Exon1 to Exon9 portions of the mouse DLL4 gene by means of CRISPR Cas9, and a DLL4 gene humanized mouse model was established. With B6 as a background mouse, a DLL4 humanized mouse model was successfully obtained.
1) Determination of human fragment substitution region and inserted human sequence
Based on the extracellular domain of human DLL4 protein and human and mouse homology comparison, the Exon1 to Exon9 portions of the human DLL4 gene replaced the Exon1 to Exon9 portions of the mouse Dll4 gene, and the sequence of the selected human DLL4 gene replacement was shown as SEQ ID No.3 (the underlined portion is the Exon sequence, and the non-underlined portion is the Intron sequence).
ATGGCGGCAGCGTCCCGGAGCGCCTCTGGCTGGGCGCTACTGCTGCTGGTGGCACTTTGGCAGCAGGT AACACGTCCCGCGCCCTCTCCGTCCCCTCTGCCGCGCTCTGGGCCTCAGCCCCGGGCACCAGCTGAGCTGACCGGT CCCCTCCCTCCTTCCCTCGGTCCCTGTGCAATAGCGCGCGGCCGGCTCCGGCGTCTTCCAGCTGCAGCTGCAGGAG TTCATCAACGAGCGCGGCGTACTGGCCAGTGGGCGGCCTTGCGAGCCCGGCTGCCGGACTTTCTTCCGCGTCTGCC TTAAGCACTTCCAGGCGGTCGTCTCGCCCGGACCCTGCACCTTCGGGACCGTCTCCACGCCGGTATTGGGCACCAA CTCCTTCGCTGTCCGGGACGACAGTAGCGGCGGGGGGCGCAACCCTCTCCAACTGCCCTTCAATTTCACCTGGCCG GTGAGCACAGCCTGGGCGCACTGGGAGGTCGCAGAAGCCGAGAGAGGAGGCGCCCTGGGACCAAAGCCCCCTCCCCAGATTTCCTTGTACACACACCCCCACCCCCAAAAAGCCCAGGATGCATTCTTTCCTGGCTCTTCCCGACTCTCTCCTGAGACTGATCCCAGAAAAGGCTCTCACCAGTCTCCGTCTTCCCAGTTTATGTCCTCCCGTCCCCAGCTCTTGGGACACGATTTTCATTACCTACCACTCTGGGGCGGTACCCTACCACCCCCTCCTCCAGTGGCTCTCCCTTACACTCTCCCGTCTCTCAACCCTCCCTCTACCGGGGGTTCTCCTCTCGCCTTCCCTGCTCAAGCGCTACACTGTGCACAGCCCCGTTATGTTGACCCGGGCGCAGTAACTGAATCCTGCAATTAGATTAATTAAACAGGCTGCCGCAAGGCACCCCCACCTCTCCCCGCTTGCTCATCTCGCCATCTCTCCGTCCCCCCACCCCCTTTCCCAGGGTACCTTCTCGCTCATCATCGAA GCTTGGCACGCGCCAGGAGACGACCTGCGGCCAGGTGAGTAGCTCGCTCCGCCACCACAGGGGGGCGACACGGCGCAGCGCCGAAAGAGTTAATCTGTTCTAGGCGGGGGAAGTGCGGGCTTGGGGGTGGGAGGCAGGACGCTTAGCTTGGCCTGGAGCTGCGCCCCGCGCTGGACGCTCGGATTCCGCTCGCTGCCTGGACTCAGAGCACAATTGCGTTTCCTGCGGGTTATTTTTGGCGTGGGAACGCGGGGAGTACGGCGGTGAGAAAGGCTGAAGCTGCCAGCGCCGCTGACGGGCCCCTTCCTGTATTTTACACCTTTCGCGAATTCCGCTCCTTTGGAAAGGGAATAATGGCTTTGGGATGTTGTTCTGACACAGAGGAAAAGGATATTTCAGCAGCACAACAATTCTCACTTTGAAAAGGAAAAAAGAAAACCATTACCCACCTCTGGAGGCAGAACCCCTGAATGGGCACCAAAGGACCCCCTGCTCCCAGGGTCCTCTCTAGCCTGGGGAGCTTTTCTTTCTTTTTCTCTTTTTTCCATTTTGACCTCTTTTCCTCTTTCCCCTCCCTATCTGCCTCCAAGACCCTGGGATATCTTAACATCCTTCTATTGTCCCCTTTTTGAATACTATCAGGCCCCCTGCACATGCACACACGTAGGGCAGCTACGTAGCGGGGCTTTGGGTCCCTCTGGCCTGTTCTTGCTGGCAGGCGGGGGTCATCTGGATAACTGGGCTGATTGGTTGGCTGATCACCATCATCACAGCCAAGAAGGACATTGGCCAGCCGTCACTGGCACCCTTGGGGACTGGCGACCCTTCCCTGACCCGACCCTCTGCCCCCTCAGAGGCCTTGCCACCAGATGCACTCATCAGCAAGATCGCCATCCAGGGCTCCCTAGCTGT GGGTCAGAACTGGTTATTGGATGAGCAAACCAGCACCCTCACAAGGCTGCGCTACTCTTACCGGGTCATCTGCAGT GACAACTACTATGGAGACAACTGCTCCCGCCTGTGCAAGAAGCGCAATGACCACTTCGGCCACTATGTGTGCCAGC CAGATGGCAACTTGTCCTGCCTGCCCGGTTGGACTGGGGAATATTGCCAACAGCGTAAGCAGTCAAGCTCCCACCTGTGTGGAAGGGGAGGGTCCCCTGAGGAAACACAGTGGAGCTTCTTGGTCACAGCTTGCCTCCCTTGAAGAGTGGGTCTGGGCCTCCTACTAGCTGGGCCTCAGGGATGCTGAGGGTGGGCTTGACCTCAGACCTCCTGTCTCTTCCCAGTGCTCCTCCCATCATGCCAAAGCCCACAAGAACCCCATCATGACATTCCATCCAGTTTGGCTTCTCCTTCCCTGTGCCATTATTTCACTTTAAGACACTCGGGGCTCCTCTGGGAGGCCAGGAGTAGGAAGAGGGCCCAGGAGAGCTAGGGGATCCCCAGGGCCAGCAGGTGAGAATGGGGCTTAAGAGTCCTTGGTATCCCAGCCTCACCCAGCTCTGTGTTCTTCCCTTAGCTATCTGTCTTTCGGGCTGTCATGAACAGAATGGCTACTGCAGCAAGCCAGCAGAGTGCCTGTGAGTAGGGGACAGGAAGTGGTGAGTGGGAGCCCTCCCTTGGCCAAGGCCTCTCACCTCACTCTGCCTCTCTCTTGTTCCCCAGCTGC CGCCCAGGCTGGCAGGGCCGGCTGTGTAACGAATGCATCCCCCACAATGGCTGTCGCCACGGCACCTGCAGCACTC CCTGGCAATGTACTTGTGATGAGGGCTGGGGAGGCCTGTTTTGTGACCAAGGTGAGTCAGGGTGAAGAGAGGGTGCAGAGGGTGCAAGAGATATGGGGCTGGGGGGTGGAAATCCGATTCGTCACCTGGATCCTTCTTACTTGGTGACTGCAGACTTGGCTTTCCCATGATCTTCCAAGGATCTTGGGTCTTTTAAGGATCTTTACAACTGGCCCAGAATGAGGCGGTGGGTCCTTCTCCAGGTGCGGCGGCAGGGGGTGGTGGAGCCAGGGTGGCTGAAAAACCCAGGGGGGTGACAAGGTCGGCAGCCTGGAGGTTGCACTCATAAATCCTAGCAAAGCCAAAGAGAGAGGGATGGCAGGCTCAGTTCCTCTTTCAACCCCGTAGTTACCTATTAACCCCCTGAGTGTTTGCTTACCTTCCAGGGCTGTTTGAGCAGCTCTCCCCTAAACAGCTGTCCGGTGGGGTGTGCCCACCGGCCACCTGAGGCTGTGGGTGAGCTGGGCCTCTGGGCGGAGTGGCATCTAACCGACTTTTCGGTGTGGGCACAAACGGCCTCCCCTGCTCTTACCTAGTTACCACCTGCCTGAACCCATGCGGTCTCTACCTGGTGTTTAGGGGTAGTCACTCTCTGGCTATACAGGGGCCTTTCAGCCCCAACCTTGGGGGAGGAGGAAGCCTTTTTTCTTGCATCCTGCTAGCCAGCTGCAGCCAGCTGCAGCTCCCATTTTCAGGATCAAATGGGTGCACCTGCTGCCCAGAGACACCGGCGCAGGCCTGGGTAGGGTGGGCAGAGAGCTTGCCAGGGTGGAAAGAAATTGCCTAGGCCCTGACTTGCTGTCAACAAGGGGCTTGGGATTCAGTCCCTGTGTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCTGTCCCTTTACTACCATCCCCACCCCAACACTCACACACCTGGTTCCTGCTCATTCTCTTCCCTCTCCACCATATTTGCTCCCAGGTGACACAGTCATATACTCATCATATGCAAACACAGCACTTGCAGGCCATATATTTACTCTGTCTGGTTCTCCCTCCCTGTCCTTCCCAAATAAAAAAACAAATACTTATATTTCAAAATACCCTTGTAACACCTCTTCCTTTAAAAAATGCCCGATTACTGCCTATGGTGGCTCTCATCTCTCCTCTACCATTTCTACCTGTTGAAATTTTATCCCTCCTTCCAGGCTTATCTCAGCTGCCCCTCCTCCATGAAGCCTTTTCTGACTTCCTCCCCGACATGTGGCCTTGCCCTCTGCTCTTCTTCCTTATCTTCATCCTACTTGGGTTGGCAGTTTGTGAGTTTCCCTGGCAGGACGTCTTCCAGTTCCAGTTGTGTTGTTTCACTTTTGGTTGACTGCACTGGTCATATGTGATTCAAGGTGCTTTAAGAAACATGATTTTCATCCTGGCTAACACAGTGAAACCCTGTCTGTATTAAAAATACAAAAGTTAGCCAGGTGTGGTGGCAGGCACCTGTAGCCCCAGCTGCTGGGAAGGCTGAGGCAGGAGAATGGCGAAGTAGAGCTTGCAGTGAGCCGAGGTCGTGCCACTGCACTCCAGCCTGAGTGACAGAGCAAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAGAAACATGATTTTAGGCTGGGTGCGATGGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGTAGGTGGATCACTTGAAGTCAGGAGTTCGAGACCATCCTGGCCATCCTGGTGAAACCCCTGTAAAAATACAAATATTAATCGGGCACAGTGGCGCATGCCTGTAATCCCAGCTACTTAGAAGGTTGAGGTATGAGAATCGCTTGAACCCGGAAGGCGAAGGTTGTAGTGAGCCTATATCACATCACTGCACTCCAGCCTGGGCGACAGAGTGAGACTCTGTTAAAAAAAAAAAAAAAAGAAGGAAAGAAAGAGAAAGAGAGAGAAAGAAAGAAAGAAAGAGAAAGAAAAAAGATTTTATTGGTGGTGGAGGAAGGATGTTTGGGCCTGGGAGACTTTGAGTTGAGGTGTCTTTGAGCCAAACATGGGGGCAAACATGGACTGCAAGGAGCCTGGAGGTGAGTGCATTCCCTGGCCCTGCTCAGCTGCTTGGTTCCTGTTTCTGCAGATCTCAACTACTGCACCCACCACTCCCCATGCAAGAATGGGGCAACGTGCTCC AACAGTGGGCAGCGAAGCTACACCTGCACCTGTCGCCCAGGCTACACTGGTGTGGACTGTGAGCTGGAGCTCAGCG AGTGTGACAGCAACCCCTGTCGCAATGGAGGCAGCTGTAAGGTGAGGCCCAGACCAGCGCAGGAAGACAGAGGTGTCAGGTGGTGTCTGGGCATCCCTAACCTAGGCAGTTAGTGGATGTACAGCCATGGACAGGCATTGTGGGCAGGTGGAGCCCAGCCTTCAGTCACACATCCCTGCCCCCCAGGGTCTGACTTTGGCCCCTTTATGGTCTCTCTCCAGGACCAGG AGGATGGCTACCACTGCCTGTGTCCTCCGGGCTACTATGGCCTGCATTGTGAACACAGCACCTTGAGCTGCGCCGA CTCCCCCTGCTTCAATGGGGGCTCCTGCCGGGAGCGCAACCAGGGGGCCAACTATGCTTGTGAATGTCCCCCCAAC TTCACCGGCTCCAACTGCGAGAAGAAAGTGGACAGGTGCACCAGCAACCCCTGTGCCAACGGTGCGTGCTGCTGCCCTGCTAACCTGGTGGACTGGCCCTGGGGCTGAGAGAGACTTCTGGTGAGGGAGGGTCAGGAGAGGAGCGAGGCATTGTCTGCCACTCTGGCCCCCCATCTGCTCTGGAGGGCGAAGAGCTTGCTTGATCAGCTGGGGGGCTGTGGAAGCGGAGCTGGTTAGTTGCACGCAGGCCTTAGGAGCAGGGGTGGTATGCACCCTGCATAGCTTCCATTCCTATTCCCATGTCAGAACCCCGTCCTGGCTGGGGTGGCCTCTGACCCTCCCCAGGAAGTCCTGAGCTGGAGAGAGGGATGTTGGAGGCTTCATGTTTCTCCTCAAAGGAGGCAGTGATTCAGTCAGAGCCCTGCTCCTGGAGGCCTCATCTTGCCCCGTGCCCAGGTAGAGCATGAGGTAGCATGAGGCATCTTGAATGTTTGCACCTTTTGAGGCACAAAGCCTGTTGGTAATCCTTGTCTATCTGGCTCCCAGGTGACCCTCTGTGAGGCAGGCAGGCAGGCAGCGCTCAGGAGCTGGAGAGGGGTGGGAAGGGCTGAGAGGGAGTCTGCTCTCTCACTGAAGCCTCTGGCACTGCCATTTCTTCATCACTGAATGGGAAACTATAATACCTGTCCTCTGTCCTTCATGTGGTTGTGAAGATGAAGTAAAACAGTCATGATTGTACTTATCCGAGCATTAACTATATACCAAACATGGGCTCTTGCCTTCATGTACCTTCCCGGCTATCCTATGAAGGGGCTAGCATTCTACTCCAGTCTAACAAATGGGGAAACTGAGGCTTAGAGACACGGTTAAGCAGCAAGTGCCAGATCTCAGGCCACAGAGTGACAGCTGAGGTCCCAACTCAAGCCTATCTGTCTGATTCTACGTTAAAGTTCTGTAAGATGCTAGTCATTTTTATACATGAGCCCACTGAGGCCGAGAGAATCAAGGTCATGCTAAACTCCAGGTCTCCTGACTCTGTGCAGTTCTCTTTGTAGTGGGCTCTGCAGGTGGAGGTAGAAGGGCCCGAACGTGTTCCTGGAATGGGGCTCCCACCCCCTGCCCCAGGGAGCTCCCAGGCTATCACTGACTTGTGTCTCATGCGTCCTCACAGGGGGACAGTGCCTGAACCGAGGTCCAAGCCGCATGTGCCGCTGCC GTCCTGGATTCACGGGCACCTACTGTGAACTCCACGTCAGCGACTGTGCCCGTAACCCTTGCGCCCACGGTGGCAC TTGCCATGACCTGGAGAATGGGCTCATGTGCACCTGCCCTGCCGGCTTCTCTGGCCGACGCTGTGAGGTGCGGACA TCCATCGATGCCTGTGCCTCGAGTCCCTGCTTCAACAGGGCCACCTGCTACACCGACCTCTCCACAGACACCTTTG TGTGCAACTGCCCTTATGGCTTTGTGGGCAGCCGCTGCGAGTTCCCCGTGGGCTTGCCGCCCAGCTTCCCCTGG（SEQ ID No.3）
2) Humanized targeting vector construction
Constructing the Exon 1-Exon 9 parts of the humanized DLL4 gene into targeting vectors, replacing the Exon 1-Exon 9 parts of the murine Dll4 gene by using the homologous recombination technology, and constructing a successful targeting vector with the sequence shown in SEQ ID No.4 (italics indicate inserted fragments, 1238bp-8143bp indicate inserted humanized DLL4 gene fragments).
1 ACGAGC CAGGAG GGAGTG GGGGTG GGGGAC AAAGAG AGAAGG CACGCG CTGGGC ACGCCC
61 TGCTGC TTGAGG TGACAC GCCTAC CAGGTG CAGAGA AGACCA GAGTCC TAGACG CTTGGC
121 CCTTTG CCTTGC TTGGAA TAGCCA GGGAAA GGTTAA GGCGTC GGATGG CTTCCC TGCGGT
181 GCTGGG GACGCG TGGAGG GTGGGC ACACAT AGGCTG GAGGCC AGCGAG GCAGGA GCTACC
241 TAAAGT CTGGAA AGGAAA GGGAGA TCCAAA TCCCCT GGTCCT GCTTTT TGCTTT CCTAGT
301 TTAAGC TTTCCC CACCTG CTAGAG GACTGT AGGTAT CTAATG CCTGGA TCAGGT GCACCG
361 CCTACG GGGACC CCTTAG AGTTTC CACCCC CTGGAC CATTCG GGAACC ACCTCA CCTCCC
421 GCCGCA TCACTG GGCTAC CCTCCT ATCCTC TGGTGG CGAGGG TCTCAG CCTTTA AGCAGA
481 CGATCT CTAAGG ACTGCT CGCCGG GCACGC GCAGAG CTGGAA GCCCAG AAGTTG GAAGAG
541 GGGCGG GGACCT GCGCCC TACTGG CTGGCT GACAGG GGGAGC GGCGGG GGCGGA GGCCCC
601 CTCCGG TGGGTG CTGGGA CTGTAG CCACTA GAGGCC TGGAGG GGAGGG GAGAGT GACCGT
661 GAGTCT GTCTGA CTGACA GGCTGC GAAGAG CAGCCA ATATAT ATAAGA AAGGCT CTGGAG
721 CAAGCA GGTTTC AGTAGC GGCGCT GCTCGC AGGCTA GGAACC CGAGGC CAAGAG CTGCAG
781 CCAAAG TCACTT GGGTGC AGTGTA CTCCCT CACTAG CCCGCT CGAGAC CCTAGG ATTTGC
841 TCCAGG ACACGT ACTTAG AGCAGC CACCGC CCAGTC GCCCTC ACCTGG ATTACC TACCGA
901 GGCATC GAGCAG CGGAGT TTTTGA GAAGGC GACAAG GGAGCA GCGTCC CGAGGG GAATCA
961 GCTTTT CAGGAA CTCGGC TGGCAG ACGGGA CTTGCG GGAGAG CGACAT CCCTAA CAAGCA
1021 GATTCG GAGTCC CGGAGT GGAGAG GACACC CCAAGG GATGGC GGCAGC GTCCCG GAGCGC
1081 CTCTGG CTGGGC GCTACT GCTGCT GGTGGC ACTTTG GCAGCA GGTAAC ACGTCC CGCGCC
1141 CTCTCC GTCCCC TCTGCC GCGCTC TGGGCC TCAGCC CCGGGC ACCAGC TGAGCT GACCGG
1201 TCCCCT CCCTCC TTCCCT CGGTCC CTGTGC AATAGC GCGCGG CCGGCT CCGGCG TCTTCC
1261 AGCTGC AGCTGC AGGAGT TCATCA ACGAGC GCGGCG TACTGG CCAGTG GGCGGC CTTGCG
1321 AGCCCG GCTGCC GGACTT TCTTCC GCGTCT GCCTTA AGCACT TCCAGG CGGTCG TCTCGC
1381 CCGGAC CCTGCA CCTTCG GGACCG TCTCCA CGCCGG TATTGG GCACCA ACTCCT TCGCTG
1441 TCCGGG ACGACA GTAGCG GCGGGG GGCGCA ACCCTC TCCAAC TGCCCT TCAATT TCACCT
1501 GGCCGG TGAGCA CAGCCT GGGCGC ACTGGG AGGTCG CAGAAG CCGAGA GAGGAG GCGCCC
1561 TGGGAC CAAAGC CCCCTC CCCAGA TTTCCT TGTACA CACACC CCCACC CCCAAA AAGCCC
1621 AGGATG CATTCT TTCCTG GCTCTT CCCGAC TCTCTC CTGAGA CTGATC CCAGAA AAGGCT
1681 CTCACC AGTCTC CGTCTT CCCAGT TTATGT CCTCCC GTCCCC AGCTCT TGGGAC ACGATT
1741 TTCATT ACCTAC CACTCT GGGGCG GTACCC TACCAC CCCCTC CTCCAG TGGCTC TCCCTT
1801 ACACTC TCCCGT CTCTCA ACCCTC CCTCTA CCGGGG GTTCTC CTCTCG CCTTCC CTGCTC
1861 AAGCGC TACACT GTGCAC AGCCCC GTTATG TTGACC CGGGCG CAGTAA CTGAAT CCTGCA
1921 ATTAGA TTAATT AAACAG GCTGCC GCAAGG CACCCC CACCTC TCCCCG CTTGCT CATCTC
1981 GCCATC TCTCCG TCCCCC CACCCC CTTTCC CAGGGT ACCTTC TCGCTC ATCATC GAAGCT
2041 TGGCAC GCGCCA GGAGAC GACCTG CGGCCA GGTGAG TAGCTC GCTCCG CCACCA CAGGGG
2101 GGCGAC ACGGCG CAGCGC CGAAAG AGTTAA TCTGTT CTAGGC GGGGGA AGTGCG GGCTTG
2161 GGGGTG GGAGGC AGGACG CTTAGC TTGGCC TGGAGC TGCGCC CCGCGC TGGACG CTCGGA
2221 TTCCGC TCGCTG CCTGGA CTCAGA GCACAA TTGCGT TTCCTG CGGGTT ATTTTT GGCGTG
2281 GGAACG CGGGGA GTACGG CGGTGA GAAAGG CTGAAG CTGCCA GCGCCG CTGACG GGCCCC
2341 TTCCTG TATTTT ACACCT TTCGCG AATTCC GCTCCT TTGGAA AGGGAA TAATGG CTTTGG
2401 GATGTT GTTCTG ACACAG AGGAAA AGGATA TTTCAG CAGCAC AACAAT TCTCAC TTTGAA
2461 AAGGAA AAAAGA AAACCA TTACCC ACCTCT GGAGGC AGAACC CCTGAA TGGGCA CCAAAG
2521 GACCCC CTGCTC CCAGGG TCCTCT CTAGCC TGGGGA GCTTTT CTTTCT TTTTCT CTTTTT
2581 TCCATT TTGACC TCTTTT CCTCTT TCCCCT CCCTAT CTGCCT CCAAGA CCCTGG GATATC
2641 TTAACA TCCTTC TATTGT CCCCTT TTTGAA TACTAT CAGGCC CCCTGC ACATGC ACACAC
2701 GTAGGG CAGCTA CGTAGC GGGGCT TTGGGT CCCTCT GGCCTG TTCTTG CTGGCA GGCGGG
2761 GGTCAT CTGGAT AACTGG GCTGAT TGGTTG GCTGAT CACCAT CATCAC AGCCAA GAAGGA
2821 CATTGG CCAGCC GTCACT GGCACC CTTGGG GACTGG CGACCC TTCCCT GACCCG ACCCTC
2881 TGCCCC CTCAGA GGCCTT GCCACC AGATGC ACTCAT CAGCAA GATCGC CATCCA GGGCTC
2941 CCTAGC TGTGGG TCAGAA CTGGTT ATTGGA TGAGCA AACCAG CACCCT CACAAG GCTGCG
3001 CTACTC TTACCG GGTCAT CTGCAG TGACAA CTACTA TGGAGA CAACTG CTCCCG CCTGTG
3061 CAAGAA GCGCAA TGACCA CTTCGG CCACTA TGTGTG CCAGCC AGATGG CAACTT GTCCTG
3121 CCTGCC CGGTTG GACTGG GGAATA TTGCCA ACAGCG TAAGCA GTCAAG CTCCCA CCTGTG
3181 TGGAAG GGGAGG GTCCCC TGAGGA AACACA GTGGAG CTTCTT GGTCAC AGCTTG CCTCCC
3241 TTGAAG AGTGGG TCTGGG CCTCCT ACTAGC TGGGCC TCAGGG ATGCTG AGGGTG GGCTTG
3301 ACCTCA GACCTC CTGTCT CTTCCC AGTGCT CCTCCC ATCATG CCAAAG CCCACA AGAACC
3361 CCATCA TGACAT TCCATC CAGTTT GGCTTC TCCTTC CCTGTG CCATTA TTTCAC TTTAAG
3421 ACACTC GGGGCT CCTCTG GGAGGC CAGGAG TAGGAA GAGGGC CCAGGA GAGCTA GGGGAT
3481 CCCCAG GGCCAG CAGGTG AGAATG GGGCTT AAGAGT CCTTGG TATCCC AGCCTC ACCCAG
3541 CTCTGT GTTCTT CCCTTA GCTATC TGTCTT TCGGGC TGTCAT GAACAG AATGGC TACTGC
3601 AGCAAG CCAGCA GAGTGC CTGTGA GTAGGG GACAGG AAGTGG TGAGTG GGAGCC CTCCCT
3661 TGGCCA AGGCCT CTCACC TCACTC TGCCTC TCTCTT GTTCCC CAGCTG CCGCCC AGGCTG
3721 GCAGGG CCGGCT GTGTAA CGAATG CATCCC CCACAA TGGCTG TCGCCA CGGCAC CTGCAG
3781 CACTCC CTGGCA ATGTAC TTGTGA TGAGGG CTGGGG AGGCCT GTTTTG TGACCA AGGTGA
3841 GTCAGG GTGAAG AGAGGG TGCAGA GGGTGC AAGAGA TATGGG GCTGGG GGGTGG AAATCC
3901 GATTCG TCACCT GGATCC TTCTTA CTTGGT GACTGC AGACTT GGCTTT CCCATG ATCTTC
3961 CAAGGA TCTTGG GTCTTT TAAGGA TCTTTA CAACTG GCCCAG AATGAG GCGGTG GGTCCT
4021 TCTCCA GGTGCG GCGGCA GGGGGT GGTGGA GCCAGG GTGGCT GAAAAA CCCAGG GGGGTG
4081 ACAAGG TCGGCA GCCTGG AGGTTG CACTCA TAAATC CTAGCA AAGCCA AAGAGA GAGGGA
4141 TGGCAG GCTCAG TTCCTC TTTCAA CCCCGT AGTTAC CTATTA ACCCCC TGAGTG TTTGCT
4201 TACCTT CCAGGG CTGTTT GAGCAG CTCTCC CCTAAA CAGCTG TCCGGT GGGGTG TGCCCA
4261 CCGGCC ACCTGA GGCTGT GGGTGA GCTGGG CCTCTG GGCGGA GTGGCA TCTAAC CGACTT
4321 TTCGGT GTGGGC ACAAAC GGCCTC CCCTGC TCTTAC CTAGTT ACCACC TGCCTG AACCCA
4381 TGCGGT CTCTAC CTGGTG TTTAGG GGTAGT CACTCT CTGGCT ATACAG GGGCCT TTCAGC
4441 CCCAAC CTTGGG GGAGGA GGAAGC CTTTTT TCTTGC ATCCTG CTAGCC AGCTGC AGCCAG
4501 CTGCAG CTCCCA TTTTCA GGATCA AATGGG TGCACC TGCTGC CCAGAG ACACCG GCGCAG
4561 GCCTGG GTAGGG TGGGCA GAGAGC TTGCCA GGGTGG AAAGAA ATTGCC TAGGCC CTGACT
4621 TGCTGT CAACAA GGGGCT TGGGAT TCAGTC CCTGTG TTGTGT GTGTGT GTGTGT GTGTGT
4681 GTGTGT GTCTGT CCCTTT ACTACC ATCCCC ACCCCA ACACTC ACACAC CTGGTT CCTGCT
4741 CATTCT CTTCCC TCTCCA CCATAT TTGCTC CCAGGT GACACA GTCATA TACTCA TCATAT
4801 GCAAAC ACAGCA CTTGCA GGCCAT ATATTT ACTCTG TCTGGT TCTCCC TCCCTG TCCTTC
4861 CCAAAT AAAAAA ACAAAT ACTTAT ATTTCA AAATAC CCTTGT AACACC TCTTCC TTTAAA
4921 AAATGC CCGATT ACTGCC TATGGT GGCTCT CATCTC TCCTCT ACCATT TCTACC TGTTGA
4981 AATTTT ATCCCT CCTTCC AGGCTT ATCTCA GCTGCC CCTCCT CCATGA AGCCTT TTCTGA
5041 CTTCCT CCCCGA CATGTG GCCTTG CCCTCT GCTCTT CTTCCT TATCTT CATCCT ACTTGG
5101 GTTGGC AGTTTG TGAGTT TCCCTG GCAGGA CGTCTT CCAGTT CCAGTT GTGTTG TTTCAC
5161 TTTTGG TTGACT GCACTG GTCATA TGTGAT TCAAGG TGCTTT AAGAAA CATGAT TTTCAT
5221 CCTGGC TAACAC AGTGAA ACCCTG TCTGTA TTAAAA ATACAA AAGTTA GCCAGG TGTGGT
5281 GGCAGG CACCTG TAGCCC CAGCTG CTGGGA AGGCTG AGGCAG GAGAAT GGCGAA GTAGAG
5341 CTTGCA GTGAGC CGAGGT CGTGCC ACTGCA CTCCAG CCTGAG TGACAG AGCAAG ACTCCG
5401 TCTCAA AAAAAA AAAAAA AAAAAA AAAAAA GAAACA TGATTT TAGGCT GGGTGC GATGGC
5461 CTGTAA TCCCAG CACTTT GGGAGG CCGAGG TAGGTG GATCAC TTGAAG TCAGGA GTTCGA
5521 GACCAT CCTGGC CATCCT GGTGAA ACCCCT GTAAAA ATACAA ATATTA ATCGGG CACAGT
5581 GGCGCA TGCCTG TAATCC CAGCTA CTTAGA AGGTTG AGGTAT GAGAAT CGCTTG AACCCG
5641 GAAGGC GAAGGT TGTAGT GAGCCT ATATCA CATCAC TGCACT CCAGCC TGGGCG ACAGAG
5701 TGAGAC TCTGTT AAAAAA AAAAAA AAAAGA AGGAAA GAAAGA GAAAGA GAGAGA AAGAAA
5761 GAAAGA AAGAGA AAGAAA AAAGAT TTTATT GGTGGT GGAGGA AGGATG TTTGGG CCTGGG
5821 AGACTT TGAGTT GAGGTG TCTTTG AGCCAA ACATGG GGGCAA ACATGG ACTGCA AGGAGC
5881 CTGGAG GTGAGT GCATTC CCTGGC CCTGCT CAGCTG CTTGGT TCCTGT TTCTGC AGATCT
5941 CAACTA CTGCAC CCACCA CTCCCC ATGCAA GAATGG GGCAAC GTGCTC CAACAG TGGGCA
6001 GCGAAG CTACAC CTGCAC CTGTCG CCCAGG CTACAC TGGTGT GGACTG TGAGCT GGAGCT
6061 CAGCGA GTGTGA CAGCAA CCCCTG TCGCAA TGGAGG CAGCTG TAAGGT GAGGCC CAGACC
6121 AGCGCA GGAAGA CAGAGG TGTCAG GTGGTG TCTGGG CATCCC TAACCT AGGCAG TTAGTG
6181 GATGTA CAGCCA TGGACA GGCATT GTGGGC AGGTGG AGCCCA GCCTTC AGTCAC ACATCC
6241 CTGCCC CCCAGG GTCTGA CTTTGG CCCCTT TATGGT CTCTCT CCAGGA CCAGGA GGATGG
6301 CTACCA CTGCCT GTGTCC TCCGGG CTACTA TGGCCT GCATTG TGAACA CAGCAC CTTGAG
6361 CTGCGC CGACTC CCCCTG CTTCAA TGGGGG CTCCTG CCGGGA GCGCAA CCAGGG GGCCAA
6421 CTATGC TTGTGA ATGTCC CCCCAA CTTCAC CGGCTC CAACTG CGAGAA GAAAGT GGACAG
6481 GTGCAC CAGCAA CCCCTG TGCCAA CGGTGC GTGCTG CTGCCC TGCTAA CCTGGT GGACTG
6541 GCCCTG GGGCTG AGAGAG ACTTCT GGTGAG GGAGGG TCAGGA GAGGAG CGAGGC ATTGTC
6601 TGCCAC TCTGGC CCCCCA TCTGCT CTGGAG GGCGAA GAGCTT GCTTGA TCAGCT GGGGGG
6661 CTGTGG AAGCGG AGCTGG TTAGTT GCACGC AGGCCT TAGGAG CAGGGG TGGTAT GCACCC
6721 TGCATA GCTTCC ATTCCT ATTCCC ATGTCA GAACCC CGTCCT GGCTGG GGTGGC CTCTGA
6781 CCCTCC CCAGGA AGTCCT GAGCTG GAGAGA GGGATG TTGGAG GCTTCA TGTTTC TCCTCA
6841 AAGGAG GCAGTG ATTCAG TCAGAG CCCTGC TCCTGG AGGCCT CATCTT GCCCCG TGCCCA
6901 GGTAGA GCATGA GGTAGC ATGAGG CATCTT GAATGT TTGCAC CTTTTG AGGCAC AAAGCC
6961 TGTTGG TAATCC TTGTCT ATCTGG CTCCCA GGTGAC CCTCTG TGAGGC AGGCAG GCAGGC
7021 AGCGCT CAGGAG CTGGAG AGGGGT GGGAAG GGCTGA GAGGGA GTCTGC TCTCTC ACTGAA
7081 GCCTCT GGCACT GCCATT TCTTCA TCACTG AATGGG AAACTA TAATAC CTGTCC TCTGTC
7141 CTTCAT GTGGTT GTGAAG ATGAAG TAAAAC AGTCAT GATTGT ACTTAT CCGAGC ATTAAC
7201 TATATA CCAAAC ATGGGC TCTTGC CTTCAT GTACCT TCCCGG CTATCC TATGAA GGGGCT
7261 AGCATT CTACTC CAGTCT AACAAA TGGGGA AACTGA GGCTTA GAGACA CGGTTA AGCAGC
7321 AAGTGC CAGATC TCAGGC CACAGA GTGACA GCTGAG GTCCCA ACTCAA GCCTAT CTGTCT
7381 GATTCT ACGTTA AAGTTC TGTAAG ATGCTA GTCATT TTTATA CATGAG CCCACT GAGGCC
7441 GAGAGA ATCAAG GTCATG CTAAAC TCCAGG TCTCCT GACTCT GTGCAG TTCTCT TTGTAG
7501 TGGGCT CTGCAG GTGGAG GTAGAA GGGCCC GAACGT GTTCCT GGAATG GGGCTC CCACCC
7561 CCTGCC CCAGGG AGCTCC CAGGCT ATCACT GACTTG TGTCTC ATGCGT CCTCAC AGGGGG
7621 ACAGTG CCTGAA CCGAGG TCCAAG CCGCAT GTGCCG CTGCCG TCCTGG ATTCAC GGGCAC
7681 CTACTG TGAACT CCACGT CAGCGA CTGTGC CCGTAA CCCTTG CGCCCA CGGTGG CACTTG
7741 CCATGA CCTGGA GAATGG GCTCAT GTGCAC CTGCCC TGCCGG CTTCTC TGGCCG ACGCTG
7801 TGAGGT GCGGAC ATCCAT CGATGC CTGTGC CTCGAG TCCCTG CTTCAA CAGGGC CACCTG
7861 CTACAC CGACCT CTCCAC AGACAC CTTTGT GTGCAA CTGCCC TTATGG CTTTGT GGGCAG
7921 CCGCTG CGAGTT CCCCGT GGGCTT GCCGCC CAGCTT CCCCTG GGTCTC GCTGGG CGTGGG
7981 GCTAGT GGTACT GCTGGT GCTCCT GGTCAT GGTGGT AGTGGC TGTGCG GCAGCT GCGGCT
8041 TCGGAG GCCCGA TGACGA GAGCAG GGAAGC CATGAA CAATCT GTCAGA CTTCCA GAAGGA
8101 CAACCT AATCCC TGCCGC CCAGCT CAAAAA CACAAA CCAGAA GAAGGA GCTGGA AGTGGA
8161 CTGTGG TCTGGA CAAGTC CAATTG TGGCAA ACTGCA GAACCA CACATT GGACTA CAATCT
8221 AGCCCC GGGACT CCTAGG ACGGGG CGGCAT GCCTGG GAAGTA TCCTCA CAGTGA CAAGAG
8281 CTTAGG AGAGAA GGTGCC ACTTCG GTTACA CAGGTA AGCCAC ACCTGG AAGCCC ATAGCT
8341 TGGTCA CAGACC CTTCCA TAGTTT GACAGG ATCTCC TAGGCT GAGTGG GAGGCT GGCATC
8401 AGGCCT TGGCAA CTTTTA ATCAAG TAAGAT TGTAGT ACTGAC AAGAAG ACACTC TAGTTA
8461 CATTTA TTTTTT TTTGTG GGGGGT GGGGTG GGGTTT TTTGAG ACAAAG TTTCTC TGTGTA
8521 GCCCTA GCTGTC CTGGAA CTCACT TTGTAG ACCAGG CTGGCC TCCAAC TCAGAA ATTCAC
8581 CTGCCT CTGCCT CCCGAG TGCTGG GATTAA AGGCGT GCGTCA CCACGC CAGGCT TCTAGT
8641 TACATT TCTATA GGGACC CAGGCA CAGTGG CACAGA CTTTGT AGCCCT ACCTAC TTAGGC
8701 TAAGAC AGGAGG ATTGCT AGTTTG ATGCTA GCCTGG GTAACA TAGCAG CAGATC ATGTCT
8761 CAAAAA CATTGA GATGGC TCAGAG AGTAAA GGCACC TGCTGC CAAGCC TGGTGA CTGAAT
8821 CTGACC TCCAGG ACCTAC ATATTA GAAGTC CTTTAA CTTCTG AGACGG TGCAGT CACACA
8881 CACACA CACACA CACACA CACACA CACACA CACACA CACACA CACACA CACTAA TTTTTA
（SEQ ID No.4）。
3) Construction of sgRNA
(1) Respectively synthesizing upstream and downstream primers of the sgRNA, wherein the purification mode of the primers is PAGE;
(2) The upstream and downstream primers of the sgRNA are respectively diluted to 100 mu mol/mu l and evenly mixed according to the proportion of 1:1, and the mixture is automatically and slowly annealed at room temperature;
(3) The double strand formed by annealing is connected with Puc57-sgRNA-NEO-Amp (Bsa I) for 1h, transformed and coated with Amp+ plates;
(4) Selecting a monoclonal, and carrying out PCR identification;
(5) Further sequencing and confirming the PCR positive monoclonal, wherein the sequencing primer is pUC57-T7-F;
(6) Using the properly sequenced clone as a template, and then using a primer to amplify the sgRNA transcribed DNA product by PCR;
(7) The sgrnas were transcribed and further purified using the transcribed DNA product of the sgrnas as templates. The constructed targeting vector is then transcribed and then reverse transcribed to obtain ssDNA donor for injection.
4) Screening of sgRNA prepared from DLL4 humanized mice
2 groups of sgrnas were designed and synthesized (5s1+3s1 and 5s2+3s2, specific sequence information is shown in table 1), the 5 'end target site and the 3' end target site were paired two by two, then the 2 pairs of sgrnas were incubated with Cas9 protein, injected into fertilized eggs for 0.5 day, cultured to blastula, and the KO positive rate of the mouse Dll4 gene was identified, thereby verifying the sgRNA cleavage activity.
The sgRNA cleavage experimental identification method comprises the following steps: the collected blasts were subjected to PCR amplification, the protocol of PCR was shown in tables 4 to 5, the amplified bands were subjected to second generation sequencing, and the results were compared with the WT bands, and the probability of mutation was counted (the identification results are shown in Table 2).
TABLE 1 sgRNA information
TABLE 2 sgRNA cleavage Activity
5) DLL4 humanized mouse model establishment
Designing and constructing ssDNA donor carrying a human sequence by using the screened sgRNA (5S2+3S2), injecting the ssDNA donor and Cas9/sgRNA system into fertilized eggs of 3 mice of 0.5d, transplanting the fertilized eggs into a pseudopregnant female mouse of 0.5d, and screening out 2 mid-target mice (F0) through gene identification after the birth of the mice.
The screened sgRNA (5S1+3s1) is designed and constructed to carry the ssDNA donor of the human sequence, the ssDNA donor and the Cas9/sgRNA system are injected into fertilized eggs of 4 mice of 0.5d and transplanted into the female mice of 0.5d pseudopregnant, but the born mice are not obtained positive F0 through genetic identification.
6) Genotyping of humanized F0 mice
The obtained rat tail genomic DNA of the F0 mouse was subjected to two-end PCR identification after mid-targeting using the two pairs of primers shown in table 3, and the PCR reaction conditions and reaction procedures are shown in tables 4 and 5, respectively. Primers GPT0X0250-01-mDll4-5tF1/GPT0X0250-01-hDLL4-5tR1 are respectively positioned outside a 5 'end homology arm and in a human fragment of ssDNA donor, if the pair of primers are amplified to generate PCR products, the target donor is effectively inserted into the 5' end of a mouse genome; GPT0X0250-01-hDLL4-3tF1/GPT0X0250-01-mDll4-3tR1 is respectively positioned in a human fragment of ssDNA donor and outside a 3 'end homology arm, and if the pair of primers are amplified to generate PCR primers, the target donor is effectively inserted into the 3' end of a mouse genome.
TABLE 3 F0 identification primers
TABLE 4 PCR reaction System
TABLE 5 PCR reaction conditions
In this test example 3F 0 mice were obtained by co-injection of the screened sgRNAs (5S2+3S2) and positive F0 mice were detected using the above-described identification protocol. As shown in the PCR electrophoresis results of FIG. 2 (WT is C57BL6JGpt genomic DNA (negative control), N is negative blank control (no template control), M is DNA Marker, TRANS 2K PLUS II band, 5000bp,3000bp,2000bp,1000bp,750bp,500bp,250bp,100 bp), the identification of both the 5 'and 3' ends of the humanized DLL4 gene of mice # 2 and # 3 is positive, tandem connection occurs, breeding can be attempted, and # 1 is negative. In addition, other batches of F0 mice are identified by the identification method, and F0 positive mice which can be bred are obtained.
And F1 is obtained by breeding positive F0 mice and background mice, the gene identification is carried out on the tail of the F1 generation, the gene identification results of the F1 generation mice are shown in a graph shown in fig. 3, the 5 'and 3' end identifications of the 6# and 9# mice humanized DLL4 genes are positive, and meanwhile, the detection of the murine source is positive, so that the obtained mice are heterozygous positive mice for correctly carrying out gene recombination. And (3) carrying out mass propagation on the F1 mice, and then carrying out inter-matching to obtain the F2 homozygous mice.
Test example 2, B6-hDLL4 humanized homozygous mouse Gene and protein expression verification
(1) Detection of B6-hDLL4 humanized homozygous mouse mRNA expression
1. Test method
Lung and thymus tissues of B6-hDLL 4F 2 homozygous mice and B6 background mice were collected for RT-PCR detection.
2. Test results
As shown in FIG. 4, the murine Dll4 mRNA was detected only in lung and thymus tissues of wild type mice, and the human DLL4 mRNA was detected only in lung and thymus tissues of homozygous B6-hDLL4 mice, indicating that B6-hDLL4 homozygous mice expressed only human DLL4 genes.
(2) Detection of B6-hDLL4 humanized homozygous mouse DLL4 protein expression
1. Test method
Lung tissue from B6-hldl 4F 2 homozygous mice and B6 background mice were collected for WB detection using human-mouse co-recognized DLL4 antibodies.
2. Test results
The results of the assay are shown in FIG. 5, and DLL4 expression was detected in both wild type mice and homozygous B6-hDLL4 mice using known human-mouse consensus anti-DLL 4 antibodies. The results described in connection with FIG. 4 demonstrate that successful expression of human DLL4 protein, but not murine Dll4 protein, was achieved in B6-hDLL4 humanized homozygous mice.
The test results show that the B6-hDLL4 mouse model is successfully constructed by replacing the humanized gene of the mouse DLL4 gene, which indicates that the model has wide application prospect in the fields of oncology, immunology and the like.
Although the method has been described in detail with respect to the steps, it will be apparent to those skilled in the art that modifications may be made to some of the parameters and aspects of the overall process within the scope of the invention. Therefore, the present invention is intended to cover all modifications, alternatives, and adaptations falling within the spirit and scope of the present invention.

Claims (10)

1. A method for constructing a DLL4 humanized mouse model, the method comprising the steps of:
(1) Constructing a targeting vector for expressing the humanized DLL4, and inserting the humanized DLL4 gene;
(2) Designing sgrnas for the Exon1 to Exon9 parts of the mouse Dll4 gene, and obtaining the sgrnas by using an in vitro transcription technology;
(3) Co-injecting or co-electrotransferring the targeting vector constructed in the step (1), the sgRNA obtained in the step (2) and the Cas9 protein into cytoplasm or nucleus of fertilized ovum of the mouse, transplanting the fertilized ovum into pseudopregnant mouse, carrying out genotype identification on pseudopregnant mouse, and screening positive F0 mouse successfully inserted into correct human fragment;
(4) F0 mice and background mice are bred to obtain F1 mice, the tail of the F1 mice is subjected to gene identification, and a DLL4 humanized mouse model is screened.
2. The construction method according to claim 1, wherein the step (1) comprises the steps of: according to the structure and function of the human DLL4 gene, the signal peptide and extracellular region of the human DLL4 gene are selected to replace the signal peptide and extracellular region sequence of the murine Dll4 gene, the amino acid sequence of the selected human DLL4 gene is shown as SEQ ID No.1, and the amino acid sequence of the replaced murine Dll4 gene is shown as SEQ ID No. 2.
3. The construction method according to claim 1, wherein the step (1) comprises the steps of: the Exon1 to Exon9 parts of the human DLL4 gene are selected to replace the Exon1 to Exon9 parts of the mouse Dll4 gene, and the sequence of the selected human DLL4 gene is shown as SEQ ID No. 3.
4. The construction method according to claim 1, wherein the sequence of the targeting vector successfully constructed in the step (1) is shown in SEQ ID No. 4.
5. The method of claim 1, wherein the sgrnas in step (2) have the gene sequences of (a) SEQ ID No.5 and SEQ ID No.7, or (b) SEQ ID No.6 and SEQ ID No.8.
6. The construction method according to claim 5, wherein the sgRNA in the step (2) has the gene sequences of SEQ ID NO.6 and SEQ ID NO.8.
7. The construction method according to claim 1, wherein the strain of mice and pseudopregnant mice provided with fertilized eggs in the step (3) is B6 mice.
8. The construction method according to claim 1, wherein the 5 '-terminal identification primer used in the genotyping of the F0 mouse in the step (3) is shown in SEQ ID NO.9 and SEQ ID NO.10, and the 3' -terminal identification primer is shown in SEQ ID NO.11 and SEQ ID NO. 12.
9. Use of the mice obtained by the construction method of any one of claims 1-8 for studying DLL4 gene related functions and mechanisms of action.
10. Use of mice obtained by the construction method of any one of claims 1-8 for screening for drugs for treating diseases associated with DLL4 genes.
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