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Transgenic animal production method and rabies animal model construction method

A technology of transgenic animals and production methods, which is applied in the field of transgenic animal production methods and rabies animal model construction, can solve problems such as raising the threshold of rabies research, and achieve the effect of improving biological safety and lowering the research threshold

Pending Publication Date: 2022-04-08
HUAZHONG UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ABSL-3 has strict requirements and regulations from architectural design and construction to laboratory personnel and environmental management, which greatly raises the threshold for rabies research
The second is the difficulty of obtaining and operating street drugs

Method used

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  • Transgenic animal production method and rabies animal model construction method
  • Transgenic animal production method and rabies animal model construction method
  • Transgenic animal production method and rabies animal model construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0068] Example 2 Transgenic mouse construction

[0069] C57BL / 6 mice were used as experimental animals.

[0070] The targeting vector was linearized, and then ES cells (Embryonic stem cells, embryonic stem cells, hereinafter referred to as ES cells) of C57BL / 6 mice were electroporated. Screen correctly recombined ES cell clones (i.e. ES cell clones that successfully target).

[0071] Screening strategy: Select the Southern blot method, and use 5'Probe (probe), 3'Probe and NeoProbe to screen ES cells at the same time, and introduce a 5' Southern restriction site between the 5' loxP site and the 3×Stop element point, there is a 3' Southern restriction site downstream of the Neo cassette, if correct recombination occurs, there will be two bands of wild type and mutant type; if not correct recombination, only the wild type band will appear.

[0072] restriction endonuclease Probe Wild type (WT) amplification results Mutant (Targeted) amplification results Hi...

Embodiment 3

[0091] Embodiment 3 Rabies animal model construction

[0092] The GT mice capable of expressing the G protein constructed in Example 2 were used as infected animals, and the G protein-deficient CVS-N2C strain RV was used as the infection strain to inoculate and infect GT mice (intranasally or intramuscularly).

[0093] Such as Figure 9 Shown, 1 is the original genotype of this virus strain, carries N, P, M, G, the coding gene of five kinds of viral proteins of L. 2 is the genotype after the strain lost the G protein, and the G protein was replaced by GFP (green fluorescent protein).

[0094] After infection, any infected cell in GT mice provides G protein to G protein-deficient CVS-N2C strain RV, restores the infection and transmission activity of street virus RV, simulates the natural infection process of RV street virus, and successfully constructs rabies animals Model.

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Abstract

The invention discloses a transgenic animal production method and a rabies animal model construction method. The exogenous G protein coding gene is inserted into the embryonic stem cell of an experimental animal, so that a transgenic animal of exogenous systemic expression G protein can be obtained. A transgenic animal is infected with G protein-deficient street virus, and G protein is supplemented for the deficient street virus by the transgenic animal, so that the rabies animal model can be obtained. The transgenic animal supplements G protein for defective RV to recover the street toxin activity, and the difficulty of obtaining, amplifying and storing the RV street toxin can be greatly reduced; after the defective RV infected in vivo leaves the animal body, the defective RV has no independent infection and transmission capability when no G protein from other sources is supplemented, so that the defective RV can be regarded as a non-infectious material, and the biological safety risk and threshold are greatly reduced. The amount of G protein provided by a transgenic animal is less than the amount of G protein expressed by RV street virus, so that the rabies progress of an animal model can be better controlled and observed compared with the rabies progress in a natural state.

Description

technical field [0001] The invention relates to the technical field of model animal research, in particular to a method for producing a transgenic animal and a method for constructing a rabies animal model. Background technique [0002] Rabies is one of the deadliest neuroviral diseases known worldwide. The vast majority of cases have been associated with dog bites in Europe, Asia, and Africa, and bat bites in the Americas. The rabies phenotypes caused by different subspecies of Rabies Virus (hereinafter referred to as RV) are different, which can be roughly divided into manic rabies and paralytic rabies. They are not the same, but the same thing is that the mortality rate is close to 100% after the onset of the disease. Tens of thousands of people die from rabies every year in the world. [0003] RV is classified in the genus Lyssavirus of the family Rhabdoviridae. The virus shell is elastic, wrapped by an envelope, and the nucleocapsid is helically symmetrical, containi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/47A01K67/027
Inventor 李鑫焱宋一舸李浩张冰李澜芳
Owner HUAZHONG UNIV OF SCI & TECH
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