Preparation method and application of humanized SIRPA genetically modified animal model

An animal model and humanized technology, applied in botany equipment and methods, biochemical equipment and methods, plant gene improvement, etc., can solve problems such as the complexity of genetic factors, reduce the risk of drug development, and speed up the research and development process , saving time and cost

Active Publication Date: 2018-09-14
BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the differences in physiology and pathology between animals and humans, coupled with the complexity of genes...

Method used

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  • Preparation method and application of humanized SIRPA genetically modified animal model
  • Preparation method and application of humanized SIRPA genetically modified animal model
  • Preparation method and application of humanized SIRPA genetically modified animal model

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Embodiment 1

[0209] Embodiment 1 sequence design

[0210] Non-human animals, such as the mouse Sirpa gene and the human SIRPA gene both contain multiple transcripts, and the sequence design of this embodiment is mainly described using one of the transcripts as an example. That is, the main part of exon No. 2 of the mouse Sirpa gene (Gene ID: 19261) (based on the transcript whose NCBI accession number is NM_007547.4 → NP_031573.2, its mRNA sequence is shown in SEQ ID NO: 1, The corresponding protein sequence is shown in SEQ ID NO: 2) with the corresponding fragment replacement of human SIRPA gene (Gene ID: 140885) (based on the transcript of NM_080792.2 → NP_542970.1 whose NCBI accession number is, its mRNA sequence is shown in SEQ ID NO : shown in 3, and the corresponding protein sequence is shown in SEQ ID NO: 4), wherein, the comparative schematic diagram of mouse Sirpa and human SIRPA gene is shown in figure 1 , the schematic diagram of the finally obtained transformed humanized mouse ...

Embodiment 2

[0216] Design and construction of embodiment 2 vector pClon-4G-SIRPA

[0217] According to the sequence design, the inventor has further designed such as image 3 The targeting scheme and the vector containing the 5' homology arm, the human SIRPA gene fragment, and the 3' homology arm are shown. Wherein the 5' homology arm (SEQ ID NO: 29) is 129607346-129608914 nucleotides of NCBI accession number NC_000068.7, and the 3' homology arm (SEQ ID NO: 30) is NCBI accession number NC_000068. 129609239-129610638 nucleotides of 7, and the human SIRPA (SEQ ID NO: 31) gene fragment is 1915110-1915433 nucleotides of NCBI accession number NC_000020.11.

[0218] The construction process of the vector is as follows: design the upstream primers for amplifying three homologous recombination fragments (LR, A, RR), the matching downstream primers and related sequences. Among them, the 5' homology arm corresponds to the LR segment, the human SIRPA gene segment corresponds to the A segment, and ...

Embodiment 3

[0229] Example 3 Verification of carrier pClon-4G-SIRPA

[0230] Randomly select 3 pClon-4G-SIRPA clones, and use 2 kinds of restriction endonucleases for enzyme digestion verification. Among them, EcoRI should appear 1371bp+5439bp, and BamHI should appear 52bp+321bp+900bp+5537bp. For enzyme digestion results, see Figure 4 , the digestion results of plasmids numbered 1, 2, and 3 were all in line with expectations, indicating that the verification results of plasmid digestion were correct. Plasmids 1 and 2 were verified to be correct by the sequencing company, and plasmid 2 was selected for subsequent experiments.

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Abstract

The invention discloses a sgRNA capable of specifically targeting Sirpa gene, a targeting vector, and a SIRPA gene humanized animal model. The invention also discloses a preparation method of an sgRNAvector of the humanized animal model, a preparation method of the humanized animal model and related application. The invention also relates to humanized genetically-engineered non-human animals, inparticular relates to genetically engineered rodents, in particular relates to genetically engineered mice, and in particular relates to a method for constructing the humanized SIRPA gene animal modeland use of the humanized SIRPA gene animal model in the field of biomedicine.

Description

technical field [0001] This application relates to the establishment method and application of a humanized genetically modified animal model, in particular, to a construction method based on a humanized SIRPA genetically modified animal model and its application in biomedicine. Background technique [0002] Immunotherapy, which activates the immune system to attack and kill cancer cells, has been an important area of ​​cancer research in recent years. At present, some drugs related to tumor immunotherapy have been used for treatment, and the drugs have been marketed and applied in multiple indications. For example, monoclonal antibodies targeting T cell co-stimulatory molecules CTLA-4, PD-1 and their ligands have been However, the average response rate of patients is low. Clinical practice has proved that the therapeutic effect of a single immunotherapy strategy is limited. In clinical practice, two or more immunotherapeutic methods generally need to be combined. The develo...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/90A01K67/027C12N15/113C12N9/22C12N5/10C07K19/00C12N15/62
CPCA01K67/0275A01K2217/07A01K2227/105A01K2267/0331A01K2267/0387C07K14/70596C12N9/22C12N15/907C12N2810/00
Inventor 沈月雷白阳张美玲黄蕤尚诚彰郭雅南
Owner BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD
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