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WO2024087014A1 - Procédé de préparation et d'utilisation d'un modèle animal épileptique - Google Patents

Procédé de préparation et d'utilisation d'un modèle animal épileptique Download PDF

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Publication number
WO2024087014A1
WO2024087014A1 PCT/CN2022/127374 CN2022127374W WO2024087014A1 WO 2024087014 A1 WO2024087014 A1 WO 2024087014A1 CN 2022127374 W CN2022127374 W CN 2022127374W WO 2024087014 A1 WO2024087014 A1 WO 2024087014A1
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WO
WIPO (PCT)
Prior art keywords
animal
preparation
ampulla
fallopian tube
pregnant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2022/127374
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English (en)
Chinese (zh)
Inventor
李苗
王虹
瑙曼·罗伯特
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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Publication date
Application filed by Shenzhen Institute of Advanced Technology of CAS filed Critical Shenzhen Institute of Advanced Technology of CAS
Priority to PCT/CN2022/127374 priority Critical patent/WO2024087014A1/fr
Publication of WO2024087014A1 publication Critical patent/WO2024087014A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo

Definitions

  • the present invention relates to the technical field of animal models, and in particular to a preparation method and application of an epileptic animal model.
  • microinjection which is to inject the gene editing complex directly into the nucleus of the fertilized egg through a microneedle
  • the other method is to electrotransfer the fertilized egg in vitro, in which the fertilized egg is immersed in the gene editing complex solution and a weak current is applied to stimulate the gene editing complex to be transferred into the fertilized egg to achieve the purpose of gene editing.
  • the existing methods have the following disadvantages: (1) The preliminary preparation work is cumbersome and requires a large number of animals, such as superovulation to obtain eggs, sperm, fertilized eggs, pseudo-pregnant animals, etc. (2) It relies on experienced technicians and expensive microinjection instruments and equipment. For example, fertilized egg in vitro culture technology, microinjection technology, male animal ligation technology, edited fertilized egg transplantation technology, etc.; (3) The microinjection method itself requires puncturing the zona pellucida and nuclear membrane structure of the fertilized egg to allow the gene editing complex to reach the fertilized egg nucleus directly, which will cause certain mechanical damage to the fertilized egg and affect the survival of the fertilized egg. Therefore, there is an urgent need for a method for preparing an epilepsy animal model that simplifies the surgical procedure, reduces animal trauma, and improves the survival efficiency of fertilized eggs.
  • the present invention proposes a method for preparing an epilepsy animal model and its application.
  • the preparation method of the present invention breaks through the technical limitation of delivering gene editing complexes to fertilized eggs in vitro, and adopts the method of delivering gene editing complexes to fertilized eggs in vivo, so as to achieve the purpose of streamlining the operation method, reducing the use of animals, and improving the efficiency of preparing transgenic animal models.
  • the present invention provides a method for preparing an epileptic animal model, comprising the following steps:
  • S3 Electrotransfer the ampulla of the fallopian tube, clamp the ampulla of the fallopian tube with electrode forceps, perform electric shock, and when the electric shock ends, return the fallopian tube to its original position and suture the muscle layer and skin layer.
  • the step S1 specifically includes: obtaining a pregnant female animal, putting the female animal in estrus and the male animal together in cages the night before, and checking the thrombus on the next morning to confirm whether the animal is pregnant.
  • the step S1 specifically includes: obtaining a pregnant female animal, putting the female animal in estrus and the male animal together at 6 pm the night before, and checking the plug at 9 am the next day to confirm whether the animal is pregnant.
  • the step S1 specifically includes: determining pregnancy as long as sperm is visible under a microscope in the form of a vaginal smear.
  • step S2 specifically includes: exposing the abdominal cavity, finding the fallopian tube, identifying the ampulla, sucking up the gene editing complex with a glass microelectrode, gently blowing into the ampulla from the end close to the ovary with a mouth pipette, and gently pushing the fallopian tube with tweezers to concentrate the liquid in the ampulla.
  • the port diameter of the glass microelectrode in step S2 is 200 ⁇ m; and the volume of the gene editing complex is 2 ⁇ l.
  • step S3 the clicking conditions in step S3 are: 50 V; 5 msec pulse; 50 msec pulse interval; 3 times; 10% decay ( ⁇ pulse orientation) as the Poring pulse.
  • step S3 the clicking conditions in step S3 are: 10V, 50 msec pulse, 50 msec pulse; 6 times, 40% decay ( ⁇ pulse orientation) as Transfer pulse.
  • the present invention also provides the use of the animal model prepared by the preparation method in screening drugs for treating epilepsy.
  • efficacy verification and drug screening of drugs for treating epilepsy can be carried out.
  • the preparation method of the present invention does not require superovulated female animals, sterilized male animals, or surrogate animals, thus reducing the number of animals used in the experimental process and complying with animal welfare.
  • the preparation method of the present invention does not require in vitro culture of fertilized eggs, and does not require transplantation of edited fertilized eggs into surrogate animals, which further simplifies the surgical procedures and reduces animal trauma. At the same time, since the operation process is greatly simplified, it is easier for experimental personnel to master.
  • the preparation method of the present invention avoids directly puncturing the fertilized egg and destroying the structure of the fertilized egg, effectively reduces the cell damage caused by direct puncture of the fertilized egg and injection, and improves the survival efficiency of the fertilized egg.
  • the preparation method of the present invention simplifies the operation process and is easier to master, thereby improving experimental efficiency, effectively shortening the time required to obtain gene-edited animals, and improving the preparation efficiency of transgenic animals.
  • FIG1 is a schematic flow diagram of the method of the present invention.
  • FIG. 2 is a sequencing peak diagram of the wild-type animal genome sequence (top) and the epilepsy animal model genome sequence (bottom).
  • Figure 3 is a comparison of the body appearance of wild-type animals (left) and epilepsy animal models (right).
  • Figure 4 is a screenshot of the video of the autonomous activities of an animal without an epileptic seizure.
  • Figure 5 is a screenshot of a video showing an animal displaying typical epileptic symptoms during an epileptic seizure.
  • S1 Obtain pregnant female animals. Put the female animals in estrus and male animals together at around 6 pm the night before. Check the plug at 9 am the next day, or use vaginal smear. If sperm can be seen under the microscope, it is determined to be pregnant.
  • S3 Electrotransfer the ampulla of the fallopian tube. Clamp the ampulla of the fallopian tube with electrode forceps and perform electric shock under the following conditions: 50V; 5 msec pulse; 50 msec pulse interval; 3 times; 10% decay ( ⁇ pulse orientation) as the Poring pulse, and 10V, 50 msec pulse, 50 msec pulse; 6 times, 40% decay ( ⁇ pulse orientation) as the Transfer pulse. After the electric shock, return the fallopian tube to its original position and suture the muscle layer and skin layer.
  • the present invention has been verified by experiments, and the scheme is feasible, and the F0 generation of transgenic rats can be obtained in as short as 40 days.
  • Figure 4 shows the autonomous activities of animals before epileptic seizures
  • Figure 5 shows typical epileptic symptoms in animals during epileptic seizures, i.e., the animals first experience convulsions in their limbs, which gradually develop into whole-body muscle convulsions, and then lose autonomous activities, fall to the ground, and lose balance.
  • the preparation method of the present invention can also be used for uterine electrotransduction stimulation of embryos, fallopian tube electrotransduction stimulation of embryos in the developmental stage, fallopian tube electrotransduction of other drugs, reagents or compounds into fertilized eggs, injection into the fallopian tube using LNP (nanoparticle carrier), electroporation transfection of developing fertilized eggs, etc.
  • LNP nanoparticle carrier
  • the present invention discloses an animal model of epilepsy and its application. It includes the following steps: S1: obtaining a pregnant female animal to confirm whether it is pregnant; S2: on the 0.7th day of pregnancy of the female animal, injecting the gene editing complex into the ampulla of the fallopian tube in vivo; S3: electrotransferring the ampulla of the fallopian tube, clamping the ampulla of the fallopian tube with electrode forceps, and performing electric shock. After the electric shock ends, the fallopian tube is returned to its original position.
  • the method of the present invention breaks through the technical limitations of the original in vitro delivery of gene editing complexes to fertilized eggs, and adopts the method of in vivo delivery of gene editing complexes to fertilized eggs, so as to achieve the purpose of streamlining the operation method, reducing the use of animals, and improving the efficiency of preparing epilepsy animal models.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Environmental Sciences (AREA)
  • Epidemiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Zoology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé de préparation et d'utilisation d'un modèle animal épileptique. Le procédé comprend les étapes suivantes consistant à : S1 : obtenir un animal femelle en gestation, et confirmer si l'animal femelle est en gestation ; S2 : au 0,7e jour de gestation de l'animal femelle, réaliser une opération d'injection d'un composé d'édition génique dans l'ampoule de la trompe de Fallope in vivo ; et S3, réaliser une électrotransfection sur l'ampoule de la trompe de Fallope, serrer l'ampoule de la trompe de Fallope à l'aide d'une pince d'électrode, réaliser une décharge électrique, et après que la décharge électrique est achevée, remettre la trompe de Fallope. Selon le procédé, la limitation technique d'un procédé d'origine pour administrer un composé d'édition génique à des ovules fécondés in vitro est surmontée, un procédé d'administration d'un composé d'édition génique à des ovules fécondés in vivo est utilisé, ce qui permet d'atteindre les objectifs de simplification du procédé opératoire, de réduction de l'utilisation d'animaux, et d'amélioration de l'efficacité de préparation de modèles animaux épileptiques.
PCT/CN2022/127374 2022-10-25 2022-10-25 Procédé de préparation et d'utilisation d'un modèle animal épileptique Ceased WO2024087014A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/127374 WO2024087014A1 (fr) 2022-10-25 2022-10-25 Procédé de préparation et d'utilisation d'un modèle animal épileptique

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/127374 WO2024087014A1 (fr) 2022-10-25 2022-10-25 Procédé de préparation et d'utilisation d'un modèle animal épileptique

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WO2024087014A1 true WO2024087014A1 (fr) 2024-05-02

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017170771A1 (fr) * 2016-04-01 2017-10-05 国立大学法人徳島大学 PROCÉDÉ D'INTRODUCTION D'UNE PROTÉINE Cas9 DANS UN ŒUF FERTILISÉ DE MAMMIFÈRE
JP2019097499A (ja) * 2017-12-05 2019-06-24 株式会社ベックス 哺乳動物の受精卵に物質を導入する方法
CN110951781A (zh) * 2019-12-18 2020-04-03 武汉大学 一种癫痫动物模型的构建方法及应用
CN111655861A (zh) * 2017-08-24 2020-09-11 内布拉斯加大学董事委员会 用于原位生殖系基因组工程的方法和组合物
US20200385753A1 (en) * 2016-12-23 2020-12-10 Institute For Basic Science Composition for base editing for animal embryo and base editing method
CN113528584A (zh) * 2020-04-21 2021-10-22 中国科学院脑科学与智能技术卓越创新中心 Stxbp1突变动物模型的构建及其应用
KR20210149459A (ko) * 2020-06-02 2021-12-09 한국과학기술연구원 카이닉산-유발 뇌전증에 대한 민감성이 증가된 마우스 및 이의 용도

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017170771A1 (fr) * 2016-04-01 2017-10-05 国立大学法人徳島大学 PROCÉDÉ D'INTRODUCTION D'UNE PROTÉINE Cas9 DANS UN ŒUF FERTILISÉ DE MAMMIFÈRE
US20200385753A1 (en) * 2016-12-23 2020-12-10 Institute For Basic Science Composition for base editing for animal embryo and base editing method
CN111655861A (zh) * 2017-08-24 2020-09-11 内布拉斯加大学董事委员会 用于原位生殖系基因组工程的方法和组合物
JP2019097499A (ja) * 2017-12-05 2019-06-24 株式会社ベックス 哺乳動物の受精卵に物質を導入する方法
CN110951781A (zh) * 2019-12-18 2020-04-03 武汉大学 一种癫痫动物模型的构建方法及应用
CN113528584A (zh) * 2020-04-21 2021-10-22 中国科学院脑科学与智能技术卓越创新中心 Stxbp1突变动物模型的构建及其应用
KR20210149459A (ko) * 2020-06-02 2021-12-09 한국과학기술연구원 카이닉산-유발 뇌전증에 대한 민감성이 증가된 마우스 및 이의 용도

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