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Method for establishing a ferret model applicable to human disease research and its application

A technology for ferrets and female ferrets, applied in the field of biology, can solve the problems of transgenic animals carrying viruses, low operability, low efficiency, etc., and achieves the effects of no virus hazard, stable and efficient ovulation, and improved efficiency.

Active Publication Date: 2018-09-28
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] However, on the one hand, as an emerging technology, CRISPR / Cas9 has never been applied to ferrets as a model animal; %, and the transgenic animals may carry the risk of the virus. In addition, the ferret superovulation program that has been reported so far is aimed at Marshall Ferret, and the superovulation efficiency for Angora Ferret is very low, and the eggs are immature and cannot be done. In vitro fertilization of ferrets cannot meet the requirements for efficient production of transgenic ferrets

Method used

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  • Method for establishing a ferret model applicable to human disease research and its application
  • Method for establishing a ferret model applicable to human disease research and its application
  • Method for establishing a ferret model applicable to human disease research and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1. CRISPR / Cas9 targeted modification gene vector construction and in vitro transcription

[0041]1) Construction of sgRNA transcription vector: For ferret Aspm (GenBank Accession No: XM_004756200), Dcx (GenBank Accession No: XM_004769082), Disc1 (GenBank Accession No: XM_013047589), specific sgRNA sequences were designed ( image 3 ), see Table 1 for the specific sequence.

[0042] Table 1

[0043]

[0044] Every two single-stranded nucleic acid sequences (Table 2) were annealed to form double-stranded DNA, and the double-stranded DNA was ligated into px330 (Addgene, 42230) vector.

[0045] Table 2. sgRNA cloning primer sequences

[0046]

[0047] 2) In vitro transcription of Cas9 and sgRNA: use the primers in Table 3 to add T7 transcripts to the transcription start sites of Cas9 and sgRNA by PCR method. The sequence of Cas9 is consistent with that in reference [2], and the PCR products are recovered and purified. In vitro transcription was performed us...

Embodiment 2

[0050] Example 2. Ovulation induction in ferrets

[0051] Choose 2-3 years old, weighing 1.5-2KG, non-estrous multiparous female mink for about 3 weeks, intraperitoneally inject 300 units of PMSG (Pregnant Mare Serum Gonadotropin) (Ningbo Sansheng Pharmaceutical), and 48 hours later intramuscularly inject the first injection of FSH ( FollitropinAlfa) (Merck Serono) 10 units, followed by continuous injection for 10 days, twice a day, 12 hours apart, 10 units each time, after the injection of FSH, continue to observe the estrus of ferrets, if estrus, intraperitoneal injection of 300 units of HCG (HumanChorionic Gonadotropin) (Merck Serono). Oocyte retrieval was performed 48 hours after HCG injection. This method is stable and efficient in ovulation, reaching 25-35 eggs per egg. It is applied in Angora Ferret, which is about 100% more efficient than the previous ovulation induction method (without mature oocytes) reported in the literature, and all of them are mature oocytes. ....

Embodiment 3

[0053] Example 3. Ferret in vitro fertilization

[0054] 1) Ovum retrieval: Obtain the oviduct of ferret by operation, use preheated HCZB culture medium (81.62mM sodium chloride, 4.83mM potassium chloride, 1.18mM potassium dihydrogen phosphate, 1.18mM magnesium sulfate, 5mM sodium bicarbonate, 1.7 mM Calcium Chloride Dihydrate, 31.3mM Sodium Lactate, 0.27mM Sodium Pyruvate, 20mM Hepes, 1mM Glutamine, 0.1mM EDTA 2Na, 5.5mM Glucose, 0.007% PVA, 1N Hydrochloric Acid) were washed out of the cumulus oocyte through the fimbria of the fallopian tube Cell complexes, digested with hyaluronidase into single oocytes without cumulus cells, placed in 38.5°C, 5% CO 2 Ready to use in IVF culture drops (Life Technologies) pre-equilibrated for 3 hours.

[0055] 2) In vitro fertilization: Select healthy male mink aged 3-4 years and weighing 2-4KG, take out the semen through the tail of the epididymis and immediately put it into IVF culture drops containing oocytes, and incubate for 3-4 hours t...

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Abstract

The invention relates to a method for establishing a ferret model capable of applying to study human disease and an application thereof, which belong to the biology field and specifically relates to a ferret ovulation induction technology and a ferret external fertilization technology. With the method for establishing the ferret model based on the technology of utilizing CRISPR / Cas9 on the above aspects, the method can be applied to study human diseases.

Description

technical field [0001] The invention belongs to the field of biology, and specifically relates to a ferret ovulation induction technology and a ferret in vitro fertilization technology, and a method for establishing a ferret model based on the technology of CRISPR / Cas9 based on the above aspects, and the method can be applied to the system Study human disease. Background technique [0002] The brain is a functional organ that controls cognition, memory, emotion and activity, and the deletion or mutation of some genes related to brain development directly leads to the occurrence of neurological diseases. The cerebral cortex of higher mammals has a structure of grooves and gyri, which greatly increases the surface area of ​​the cerebral cortex. Studies have shown that this is closely related to brain functions such as advanced cognition. [0003] As a new type of experimental animal, ferrets have been widely used in the research of respiratory diseases, etc., but they have no...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071A01K67/02C12N5/073C12N15/89
CPCA01K67/02C12N5/0603C12N15/89
Inventor 王晓群高绍荣吴倩寇朝辉尹崇海
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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