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Somatostatin gene defective immunodeficient mice, preparation method and application thereof

A technology of immunodeficiency mice and somatostatin, which is applied in the field of genetically engineered animals, can solve the problems of long modeling time and low success rate of modeling

Pending Publication Date: 2019-08-13
南京普恩瑞生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned immunodeficient mice (NOD-Prkdcscid IL2rgtm1 / Bcgen) still have the problem of long modeling time and low modeling success rate in the process of human tumor cell or tissue transplantation (Gu Q, Zhang B, Sun H, et al. Genomic characterization of a large panel of patient-derived hepatocellular carcinomaaxenograft tumor models for preclinical development. Oncotarget. 2015;6(24):20160-20176. and Zhu Y, Tian T, Li Z, et al. Establishment and characterization of patient-derived tumor xenograft using gastroscopic biopsies in gastric cancer. Scientific Reports. 2015;5:8542. doi:10.1038 / srep08542. etc.), thus limiting the use of this animal model in tumor drug screening, cancer treatment methods and clinical research, etc. wide application of

Method used

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  • Somatostatin gene defective immunodeficient mice, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1. CRISPR / Cas9 system construction

[0045] 1). Design of specific targeting sequence for mouse Sst gene

[0046] According to the principle of CRISPRE-Cas9 targeting, the Sst gene of PNDG mice was identified with specific targeting DNA sequence. Specifically, the signal peptide region of the first exon of the Sst gene is used as the target ( figure 1 ), design a guide RNA that specifically recognizes the Sst gene. In the first exon of the mouse Sst gene, the sequence of (N20) NGG was selected, and the BLAST (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) method was applied to the whole genome of the mouse. In the sequence, specificity verification is performed. If other genomic sequences are found to match the last 12 bases of the GG(N)18NGG sequence and the PAM sequence, it is considered that the sequence has potential off-target effects, that is, the target sequence is discarded and the design is redesigned.

[0047] According to the above principles, the inventors use...

Embodiment 2

[0066] Example 2. Construction of Sst gene knockout mice

[0067] The PNDG mice used came to the Medical Laboratory Animal Center of Nanjing Medical University (No. 101, Longmian Avenue, Jiangning District, Nanjing), and were raised at SPF level, keeping the ambient temperature 22-26°C, humidity 50-60%, and photoperiod 7:00 -19:00 light, 19:00-7:00 darkness. All experimental procedures of the gene knockout mouse model have been reviewed by the Experimental Animal Ethics Committee of Nanjing Medical University.

[0068] 1). Collection and culture of mouse fertilized eggs

[0069] 6-week-old female NDG mice were selected to induce superovulation. Under a dissecting microscope, the ampulla with enlarged fallopian tubes was torn, and the deposit containing eggs was discharged, and the mouse eggs were covered with droplets of culture fluid with paraffin oil. After culturing for 0.5 hours, add 10μl of NDG capacitated sperm, in vitro fertilization culture, culture temperature 37.0℃, CO 2 ...

Embodiment 3

[0088] Example 3. Establishment of tumor model

[0089] The SST-PNDG mouse of the present invention is used to construct an animal model of gastric lymphoma, which shows the advantages of its growth rate in the mouse body and the modeling time.

[0090] 1). Experimental materials

[0091] Tumor tissue: Provide fresh tumor tissue suspected of gastric cancer. The tumor tissue is stored in tissue preservation solution and transported at 4°C.

[0092] 2). Experimental animals

[0093] • Breed: SMST-PNDG mouse of the present invention

[0094] •Age: 5-6 weeks old

[0095] • Feeding environment: SPF laboratory animal room of Nanjing Medical University Medical Laboratory Animal Center

[0096] • Laboratory temperature: 22℃-25℃

[0097] •Relative humidity: 40%-70%

[0098] • Relative air pressure: 10 Pa -20 Pa

[0099] • Air exchange frequency: (10-15) times / hour

[0100] •Light cycle: 12 hours of light (8:00-20:00), 12 hours of darkness (20:00-8:00)

[0101] • Animal breeding: 5 animals / cage.

[0102] ...

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Abstract

The invention relates to somatostatin gene defective immunodeficient mice, a preparation method and application thereof, in particular to a method for building somatostatin gene knocked-out SST-PNDG somatostatin combined immunodeficient mice on NOD-Prkdc and IL2rg- / -immunodeficient mice (PNDG mice), SST-PNDG somatostatin immunodeficient mice obtained thereby and application thereof. The mice can be used for biomedicine, pharmacy and clinical researches like heterograft, tumor growth and drug screening.

Description

Technical field [0001] The invention relates to the field of genetically engineered animals. The invention relates to a method for knocking out the somatostatin (SST) Sst gene in immunodeficiency mice, the immunodeficiency mice obtained therefrom, and uses thereof. More specifically, the present invention relates to the construction of a new somatostatin Sst gene knockout SST-PNDG somatostatin combined immunodeficiency mouse based on NOD-Prkdc, IL2rg- / - immunodeficient mice (PNDG mice) and use. Background technique [0002] Immunodeficiency mouse models have been widely used in biomedical research, including immunology, oncology, and cell biology. Among them, xenogeneic tumor transplantation models of immunodeficient mice have been commonly used to evaluate tumor treatment methods and screen clinical treatment plans. [0003] At present, the internationally recognized immunodeficiency mice (NOD-Prkdcscid IL2rgtm1 / Bcgen) with the highest degree of immunodeficiency and suitable fo...

Claims

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Application Information

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IPC IPC(8): A01K67/027C12N15/85A61K49/00
CPCA01K67/0276C07K7/08C12N15/8509A61K49/0008A01K2227/105A01K2217/075A01K2267/0306A01K2267/0362A01K2267/0337
Inventor D.陈朱燕萍郝艳鹏
Owner 南京普恩瑞生物科技有限公司
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