Reverse cross-multistage nerve tracing method

A nerve and tracer technology, applied in the field of virus neurotracer research, can solve problems such as weak PRV labeling efficiency and labeling effect, inability to infect primates, biosafety issues, etc., to achieve labeling efficiency and excellent effect, The effect of convenient subsequent transformation and short length

Pending Publication Date: 2022-03-18
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a reverse multi-level nerve tracing method to solve the biological safety problems caused by RV labeling, as well as the problems of PRV labeling efficiency and labeling effect are weak, and can not infect primates

Method used

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  • Reverse cross-multistage nerve tracing method
  • Reverse cross-multistage nerve tracing method
  • Reverse cross-multistage nerve tracing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0058] Example 2 Transgenic mice construction

[0059] C57BL / 6 mice as experimental animals.

[0060] The target carrier is primarily subjected to EMBRYONIC STEM Cell, embryonic stem cells, and ES cells below. Screening the correct recombinant ES cell clone (ie, the Successful ES cell clone).

[0061] Screening Policy: Select the Southern Blot method while using 5'Probe (probe), 3'Probe and Neoprobe for ES cell screening, 5 'end SoutherN enzyme cutting between 5' end LOXP sites and 3 × STOP elements Point, there is a 3 'side Southern enzyme sink downstream of Neo Cassette. If the correct recombination occurs, there will be two strips of wild-type and mutant types; if it is not correct, there will only be wild-type strips.

[0062] Restricted endonuclease Probe Wild-type (WT) amplification results Targeted amplification results HindIII 5’ 4.4KB 5.5KB EcorV 3’ 11.5kb 9.5kb XbaI 3'neo - 5.7kb

[0063] When the NEO gene is integrated into eukaryo...

Embodiment 3

[0082] Example 3 Transgenic animals and G protein lack of rabies virus binding to neural trace

[0083] A gt mouse which was capable of expressing a G protein as in Example 2 was an infected animal, and the GT rats were inoculated (drip nose or muscle injection) using a G protein defective CVS-N2C strain RV.

[0084] Such as Figure 9 As shown, 1 is the original genotype of the strain, carrying the five viral proteins of N, P, M, G, and L. 2 is a genotype after the lack of G protein of the strain, and the G protein is replaced by GFP (green fluorescent protein).

[0085] After infection, any one of the cells infected by the gt mouse is a G protein defective CVS-N2C strain RV, which restores the infection of street toxic RV, and simulates the natural infection process of RV downtown, you can pass G. The fluorescent gene expression in the protein defective strain itself observes the infection and dissemination of neurons of transgenic animals that provide G protein.

Embodiment 4

[0086] Example 4 Nerve tracer using a G protein defective RV

[0087] Using SAD-B19- Δg-GFP RV of the G-protein defective (ΔG) as a tracery virus tool, injecting the strand of the strand of the gt mouse described in Example 2, the viral titer is 2 * 10 ^ 8pfu / ml, the injection volume is 1 μl.

[0088] After 24 days, the gt mouse was perfused, and the polymethylene formaldehyde was fixed to take the brain tissue, dehydrated, and finally the topic slices of the brain sample were treated. It was observed whether the brain was observed by the G protein lack of RV trails. Virus infection and tag.

[0089] Because the virus tool genome carries the expression gene GFP of the green fluorescent protein, the infected neurons will be labeled with green fluorescent. According to the experimental results, we have found that the G protein-deficient RV trace virus tool that cannot be spread and infected in wild murine can be infected with muscle infection and spread back to the brain, infected...

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Abstract

The invention discloses a reverse cross-multistage nerve tracing method, which comprises the following steps of: constructing a transgenic animal capable of expressing G protein of rabies virus, infecting the transgenic animal by using a defective rabies virus strain losing the G protein, and providing the G protein for the defective rabies virus strain losing the G protein by using the infected cells of the transgenic animal. The infection transmission activity is recovered, and the purpose of reverse cross-multistage nerve tracing is achieved. The method is more excellent in neuron marking efficiency and effect, and compared with PRV neurons, the neuron toxicity is smaller, the genome length is shorter, and primate animals can be infected, so that subsequent research on neuron morphology, namely functions, is more facilitated, and the requirements of subsequent brain connection group research are better met.

Description

Technical field [0001] The present invention relates to the field of neurological trace research using virals, and in particular, to a method of reverse spanning multi-stage nerve traces using a G protein defective rabies. Background technique [0002] Rabies (Rabies) is one of the most deadly neurological viral diseases worldwide. Most cases produce dog bite in Europe, Asia and Africa and the bat bite in the Americas. Different subspecies of rabies viruses (Rabies Virus, the following referred to as RV) is different, which is roughly divided into mad rabies and paralysis rabies. After being bitten, according to the difference between RV subspecies and the size, incidence Nor like the same, the same is that there is a hundred percent of mortality after the onset, and there are hundreds of people who have died every year in rabies. [0003] RV is classified in Rabdoviridae rabies virus (Lysavirus). The virus housing is spring-shaped, the envelope package, the nuclear shell spiral ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/47C12N15/65A01K67/027
CPCC12N15/8509C12N15/65C07K14/005A01K67/0278A01K67/027C12N2800/107C12N2800/80C12N2760/20122A01K2207/15A01K2207/20A01K2217/072A01K2227/105A01K2227/106A01K2227/10A01K2267/0337
Inventor 李鑫焱宋一舸李浩张冰李澜芳
Owner HUAZHONG UNIV OF SCI & TECH
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