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WO2024087014A1 - 一种癫痫动物模型的制备方法及应用 - Google Patents

一种癫痫动物模型的制备方法及应用 Download PDF

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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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animal
preparation
ampulla
fallopian tube
pregnant
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French (fr)
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李苗
王虹
瑙曼·罗伯特
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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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

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  • 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

提供了一种癫痫动物模型及应用。包括如下步骤:S1:获取受孕雌性动物，确认是否受孕；S2:在雌性动物受孕第0.7天，实行体内输卵管壶腹部注射基因编辑复合物的操作；S3:电转输卵管壶腹部，用电极镊夹持输卵管壶腹部，电击，电击结束，归位输卵管。所述方法突破了原有的以体外方式递送基因编辑复合物至受精卵的技术局限，采取体内递送基因编辑复合物至受精卵的方式，达到精简操作方法、减少动物使用、提高癫痫动物模型制备效率的目的。

Description

一种癫痫动物模型的制备方法及应用 技术领域
本发明涉及动物模型技术领域，特别涉及一种癫痫动物模型的制备方法及应用。
背景技术
在癫痫动物模型的制备中，如何有效递送基因编辑复合物至受精卵，使基因编辑复合物发挥编辑功能，是癫痫动物模型能否成功制备的重要因素。以往技术大部分采用体外递送的方式，如显微注射、体外电转受精卵等，这些体外递送的方式牵涉到多个步骤，如超数排卵获取雌性动物卵子、获取雄性动物精子、体外受精获取受精卵、受精卵体外培养、体外递送基因编辑复合物至受精卵、获取假孕受体雌性动物、移植已编辑的受精卵至假孕受体动物等，这些步骤不仅操作繁琐，且需要经验丰富的操作人员，做大量工作。
现有癫痫动物模型的制备中体外递送基因编辑复合物至受精卵的方式主要分为两种，一种是采取显微注射，即通过显微针头注射基因编辑复合物直达受精卵核；另一种方法采取受精卵体外电转的方式，使受精卵浸润在基因编辑复合物溶液中并施加弱电流，刺激基因编辑复合物转入受精卵，以达到基因编辑的目的。
以显微注射方法为对照：现有方法有以下几点弊端：(1)前期准备工作繁琐，需要大量的动物，如超数排卵获取卵子、精子、受精卵、假孕动物等。(2)依赖经验丰富的技术人员以及价格高昂的显微注射仪器设备。比如受精卵体外培养技术、显微注射技术、雄性动物结扎技术、已编辑受精卵移植技术等；(3)显微注射方法本身需要刺破受精卵透明带和核膜结构，才可使基因编辑复合物直达受精卵核，因此会对受精卵产生一定机械 损伤，影响受精卵存活。因此目前亟需一种精简手术程序、减少动物创伤、提高受精卵存活效率的癫痫动物模型制备方法。
发明内容
针对现有技术中的缺陷，本发明提出了一种癫痫动物模型的制备方法及应用。本发明的制备方法突破了原有的以体外方式递送基因编辑复合物至受精卵的技术局限，采取体内递送基因编辑复合物至受精卵的方式，达到精简操作方法、减少动物使用、提高转基因动物模型制备效率的目的。
本发明提供一种癫痫动物模型的制备方法，包括如下步骤：
S1：获取受孕雌性动物，确认是否受孕；
S2：在雌性动物受孕第0.7天，实行体内输卵管壶腹部注射基因编辑复合物的操作；
S3：电转输卵管壶腹部，用电极镊夹持输卵管壶腹部，电击，电击结束，归位输卵管，缝合肌肉层和皮肤层。
进一步的，所述步骤S1具体包括：获取受孕雌性动物，发情期雌性动物和雄性动物提前一天晚上合笼，第二天上午检栓确认是否受孕。
进一步的，所述步骤S1具体包括：获取受孕雌性动物，发情期雌性动物和雄性动物提前一天晚上6点合笼，第二天上午9点检栓确认是否受孕。
进一步的，所述步骤S1具体包括：以阴道涂片的方式，以显微镜下可见精子即判定为受孕。
进一步的，所述步骤S2具体包括：暴露腹腔，找到输卵管，辨别出壶腹部，用玻璃微电极吸取基因编辑复合物，从靠近卵巢一端，用口吸管轻轻吹入壶腹部，用镊子轻轻推动输卵管，使液体集中于壶腹部。
进一步的，所述步骤S2中所述玻璃微电极的端口直径为200μm；所述基因编辑复合物的体积为2μl。
进一步的，所述步骤S3中所述点击的条件为：以50V；5 msec pulse；50 msec pulse interval；3 times；10％decay(±pulse orientation)做为Poring pulse。
进一步的，所述步骤S3中所述点击的条件为：以10V,50 msec pulse,50 msec pulse；6 times,40％decay(±pulse orientation)做为Transfer pulse。
本发明还提供所述的制备方法制备得到的动物模型在筛选治疗癫痫药物中的应用。在上述技术方案中，基于癫痫动物模型，可以进行治疗癫痫药物的药效验证、药物筛选。
综上，与现有技术相比，本发明达到了以下技术效果：
1、本发明的制备方法不需要超数排卵雌性动物，结扎雄性动物，不需要代孕动物，减少实验过程动物使用数量，符合动物福利。
2、本发明的制备方法不需要体外培养受精卵，不需要移植已编辑受精卵至代孕动物，进一步精简手术程序，减少动物创伤；同时由于极大简化了操作流程，因此更容易被实验人员掌握。
3、本发明的制备方法避免了直接刺破受精卵，破坏受精卵结构，有效降低了直接刺破受精卵注射所带来的细胞损伤，提高受精卵存活效率。
4、本发明的制备方法简化操作流程，更容易被掌握，因此可提高实验效率，有效缩短获取基因编辑动物所需时间，提高转基因动物的制备效率。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本发明的方法的流程示意图。
图2为野生型动物基因组序列(上)和癫痫动物模型基因组序列(下)的测序峰图。
图3为对比野生型动物(左)和癫痫动物模型(右)的体型外观结果。
图4为癫痫未发作动物自主活动视频截图。
图5为癫痫发作期动物出现典型癫痫症状的视频截图。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
实施例
本发明技术方案由以下步骤组成：
S1：获取受孕雌性动物，发情期雌性动物和雄性动物提前一天晚上6点左右合笼，第二天上午9点检栓，或者以阴道涂片的方式，以镜下检可见精子即判定为受孕。
S2：在雌性动物受孕第0.7天，即下午4点左右实行体内输卵管壶腹部注射基因编辑复合物的操作。暴露腹腔，找到输卵管，辨别出壶腹部，用端口直径约200μm的玻璃微电极吸取2μl基因编辑复合物，从靠近卵巢一端，用口吸管轻轻吹入壶腹部，用镊子轻轻推动输卵管，使液体集中于壶腹部。
S3：电转输卵管壶腹部，用电极镊夹持输卵管壶腹部，用以下条件电击：以50V；5 msec pulse；50 msec pulse interval；3 times；10％decay(±pulse orientation)做为Poring pulse，以10V,50 msec pulse,50 msec pulse；6 times,40％decay(±pulse orientation)做为Transfer pulse。电击结束，归 位输卵管，缝合肌肉层和皮肤层。
本发明已经经过实验验证，方案可行，最短40天即可获取转基因大鼠F0代。
图2的基因组测序结果显示，癫痫动物模型第19号外显子中的两个碱基被删除，说明基因编辑成功。
图3结果显示野生型动物(左)和癫痫动物模型(右)，出生后15天体型外观表现出差异(放大倍数相同)，癫痫动物模型的体型明显偏小。
图4显示癫痫未发作动物自主活动，图5显示癫痫发作期动物出现典型癫痫症状，即动物先出现四肢抽搐现象，逐渐发展为全身肌肉抽搐，并失去自主活动，倒地，身体失去平衡。
除此之外，本发明的制备方法还可以用于子宫电转刺激胚胎、输卵管电转刺激发育阶段的胚胎、输卵管电转其他药物、试剂或者化合物入受精卵、利用LNP(纳米颗粒载体)注射入输卵管、电击转染发育中的受精卵等。
综合以上实施例的结果，本发明公开了一种癫痫动物模型及应用。包括如下步骤：S1：获取受孕雌性动物，确认是否受孕；S2：在雌性动物受孕第0.7天，实行体内输卵管壶腹部注射基因编辑复合物的操作；S3：电转输卵管壶腹部，用电极镊夹持输卵管壶腹部，电击，电击结束，归位输卵管。本发明的方法突破了原有的以体外方式递送基因编辑复合物至受精卵的技术局限，采取体内递送基因编辑复合物至受精卵的方式，达到精简操作方法、减少动物使用、提高癫痫动物模型制备效率的目的。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (9)

  1. 一种癫痫动物模型的制备方法，其特征在于，包括如下步骤：
    S1：获取受孕雌性动物，确认是否受孕；
    S2：在雌性动物受孕第0.7天，实行体内输卵管壶腹部注射基因编辑复合物的操作；
    S3：电转输卵管壶腹部，用电极镊夹持输卵管壶腹部，电击，电击结束，归位输卵管。
  2. 根据权利要求1所述的递送方法，其特征在于，所述步骤S1具体包括：获取受孕雌性动物，发情期雌性动物和雄性动物提前一天晚上合笼，第二天上午检栓确认是否受孕。
  3. 根据权利要求2所述的制备方法，其特征在于，所述步骤S1具体包括：获取受孕雌性动物，发情期雌性动物和雄性动物提前一天晚上6点合笼，第二天上午9点检栓确认是否受孕。
  4. 根据权利要求1所述的制备方法，其特征在于，所述步骤S1具体包括：以阴道涂片的方式，以显微镜下可见精子即判定为受孕。
  5. 根据权利要求1所述的制备方法，其特征在于，所述步骤S2具体包括：暴露腹腔，找到输卵管，辨别出壶腹部，用玻璃微电极吸取基因编辑复合物，从靠近卵巢一端，用口吸管轻轻吹入壶腹部，用镊子轻轻推动输卵管，使液体集中于壶腹部。
  6. 根据权利要求5所述的制备方法，其特征在于，所述步骤S2中所述玻璃微电极的端口直径为200μm；所述基因编辑复合物的体积为2μl。
  7. 根据权利要求1所述的制备方法，其特征在于，所述步骤S3中所述点击的条件为：以50V；5msec pulse；50msec pulse interval；3times；10％decay做为Poring pulse。
  8. 根据权利要求1所述的制备方法，其特征在于，所述步骤S3中所 述点击的条件为：以10V,50msec pulse,50msec pulse；6times,40％decay做为Transfer pulse。
  9. 权利要求1～8任一项所述的制备方法制备得到的动物模型在筛选治疗癫痫药物中的应用。
PCT/CN2022/127374 2022-10-25 2022-10-25 一种癫痫动物模型的制备方法及应用 Ceased WO2024087014A1 (zh)

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