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Construction method of Nf1 gene knockout animal model

A technology for constructing methods and animal models, applied in the field of gene editing, can solve the problems of difficult operation, low scalability, and high technical difficulty, and achieve the effect of good controllability, easy operation, and clinical characteristics.

Pending Publication Date: 2022-01-21
SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional Cre mouse technology currently used is relatively difficult to operate and has relatively low scalability. In recent years, CRISPR technology has been developed. Compared with traditional genome editing tools, it is easier to operate, cost-effective, and has stronger scalability. , but there is no relevant report on tumorigenesis in NF1 disease plexiform neurofibroma mouse model
At the same time, there are no reports of successful modeling of related models including traditional Cre gene-edited mice and CRISPR gene-edited mice in China.
[0005] To sum up, there is no NF1-related model established in China, and its technical difficulty is high, which is one of the important constraints affecting the improvement of NF1-related research in my country

Method used

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  • Construction method of Nf1 gene knockout animal model
  • Construction method of Nf1 gene knockout animal model
  • Construction method of Nf1 gene knockout animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, obtaining gRNA

[0039] The method for obtaining the gRNA directed at the Nf1 gene of the present embodiment is as follows:

[0040] (1) Selection of cells used for gene targeting: Schwann cells of mice with C57BL / 6 genetic background;

[0041] (2) Design the gRNA target sequence for the Nf1 gene and use it to prepare gRNA primers: Primer01 is shown in SEQ ID NO.1 or SEQ ID NO.2, and Primer02 is shown in SEQ ID NO.3. Centrifuge Primer01 and Primer02 at 13,000 rpm for 1 min. After completion, add 75 μL ddH2O to the Primer01 tube, add 256 μL ddH2O to the Primer02 tube, and directly stand at room temperature for later use;

[0042] (3) Prepare a mixed system in a PCR tube according to the following system:

[0043]

[0044] ( Max DNA Polymerase (Cat. No.: R045A)

[0045] (4) 150 μL system was divided into six tubes, each tube was 25 μL. The PCR reaction was carried out in a PCR machine, and the program was as follows: 98°C, 2min denaturation, 98°C fo...

Embodiment 2

[0048] Embodiment two, obtain Cas9 mRNA

[0049] In this example, Cas9 mRNA was obtained by in vitro transcription. The specific method is as follows:

[0050] (1) 10 μg of plasmid pX-T7 (Cas9), 5 μL of XbaI enzyme, and 100 μL of the total system; linearized with XbaI, and reacted at 37°C for 2 hours;

[0051] (2) Add 10 μL ammonium acetate, mix well, add 200 μL absolute ethanol, mix well, centrifuge at 13000 rpm for 5 min, discard the supernatant, centrifuge at 13000 rpm for 1 min, blot the residual liquid, add 20 μL nuclease-free water;

[0052] (3) In vitro transcription using mMESSAGE mMACHINE T7 ULTRA Kit (Invitrogen, AM1345);

[0053] (4) Use MEGAclear TM Transcription Clean-Up Kit (Invitrogen, AM1908) was purified and stored at -80°C.

Embodiment 3

[0054] Example 3, construction of homologous recombination vector

[0055] The construction method of the homologous recombination vector of this embodiment is as follows:

[0056] (1) Use the sequences shown in SEQ ID NO.8 and SEQ ID NO.9 as primers, use the wild-type mouse genome as a template, and obtain the target fragment of the 5' homology arm by PCR amplification; use such as SEQ ID NO. The sequences shown in ID NO.10 and SEQ ID NO.11 are used as primers, and the wild-type mouse genome is used as a template to perform PCR amplification to obtain the target fragment of the flox region; using such as SEQ ID NO.12 and SEQ ID NO. The sequence shown in 13 was used as a primer, and the wild-type mouse genome was used as a template to obtain the target fragment of the 3' homology arm by PCR amplification. The PCR primers are shown in Table 1:

[0057] Table 1

[0058]

[0059] (2) Digest the plasmid pBR-322 with BamHI and HindIII to obtain the linearized pBR-322 backbone ...

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Abstract

The invention discloses a construction method of an Nf1 gene knockout animal model. The construction method comprises the following steps of designing gRNA for identifying an Nf1 gene; carrying out in vitro transcription to obtain Cas9mRNA and gRNA (guide Ribonucleic Acid); constructing a homologous recombinant vector; microinjecting the Cas9mRNA, the gRNA and the homologous recombinant vector into fertilized eggs of animals, and obtaining F0-generation animals; and mating a positive F0-generation animal with a wild-type animal to obtain an F1-generation animal, namely the Nf1 gene knockout animal model. The animal model is the first successfully tumorigenic I-type neurofibroma transgenic animal model in China, provides a stable and reliable in-vivo model platform for research of the occurrence and development mechanism of I-type neurofibroma and exploration of therapeutic schedules such as targeted therapy, and makes up for the deficiency of lack of related models at present.

Description

technical field [0001] This application relates to the technical field of gene editing, in particular to a method for constructing an Nf1 gene knockout animal model. Background technique [0002] Neurofibromatosis Type 1 (NF1) is an autosomal dominant genetic disease caused by Nf1 gene mutation. It is one of the most common skin and soft tissue neoplastic diseases in plastic surgery, with an incidence of about 1:3500. . Nf1 mutation or deletion leads to inactivation of neurofibromin, activated Ras cannot be hydrolyzed into inactivated Ras, and continuous abnormal activation of RAS pathway leads to abnormal cell proliferation. The main manifestations of patients are benign neurofibromas with abnormal proliferation of Schwann cells, including cutaneous neurofibromas and plexiform neurofibromas. [0003] Because Nf1 mutations begin at the fertilized egg stage, the lesions can involve multiple systems such as skin, bones, blood vessels, and nerves, with various clinical manife...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12A01K67/027
CPCC12N15/8509C07K14/47A01K67/0276A01K2217/075A01K2227/105A01K2267/0331
Inventor 王智超李青峰魏澄江王薇顾熠辉
Owner SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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