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Method and application for fast retrograde transsynaptic labeling of nerve cells

A neural and purposeful technology, applied in the field of rapid retrograde transsynaptic labeling of nerve cells, can solve the problems of limited double-stranded AAV load, long experimental cycle, and large size of auxiliary gene RVG

Active Publication Date: 2019-01-25
WUHAN INST OF PHYSICS & MATHEMATICS CHINESE ACADEMY OF SCI
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Problems solved by technology

[0005] However, the existing technology uses defective RV combined with single-chain AAV helper virus to achieve retrograde transmonosynaptic labeling. It is necessary to inject the AAV helper virus in the initial brain area 2-3 weeks in advance, so that the exogenous gene is expressed in large quantities before injecting the defect. Type RV and expressed for a week, this method requires a relatively long experimental period, about one month
However, the load capacity of double-stranded AAV is limited, and the size of the helper gene RVG is relatively large, which is 1575bp. Can it be loaded onto double-stranded AAV and successfully rescue the virus and achieve specific expression in cre transgenic mice and helper-deficient The function of RV across synapses has not been reported

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  • Method and application for fast retrograde transsynaptic labeling of nerve cells
  • Method and application for fast retrograde transsynaptic labeling of nerve cells
  • Method and application for fast retrograde transsynaptic labeling of nerve cells

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Embodiment 1

[0033] A method for rapidly retrograde transsynaptic labeling of nerve cells, comprising the steps of:

[0034] 1. Construction of helper virus vector and virus packaging

[0035] 1. Construction of helper virus vector

[0036] SCAAV-CMV-EGFP (Wang Z et al., Gene Ther.2003, 10(26): 2105-11.) was recovered by double cutting with MluI (ACGCGT) and HindIII to obtain SCAAV (M / H); AAV-hSyn -DIO-EYFP (purchased from Wuhan Privy Brain Science and Technology Co., Ltd.) was double-cut and recovered with MluI (ACGCGT) and HindIII to obtain hSyn-DIO-EYFP (M / H); SCAAV (M / H) vector and hSyn-DIO -EYFP(M / H) fragments were ligated with T4 ligase overnight at 16°C, transformed into StbI3 competent for colony PCR identification, placed in LB medium, cultured overnight at 30°C, extracted plasmids for enzyme digestion verification and sequencing, SCAAV-hSyn-DIO-EYFP was obtained.

[0037]SCAAV-hSyn-DIO-EYFP was double-cut with NheI / AscI to recover the vector to obtain SCAAV-hSyn-DIO(N / A), AAV-...

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Abstract

The invention belongs to the construction field of auxiliary viruses, and discloses a method and an application of fast retrograde transsynaptic labeling of nerve cells. The invention uses double-stranded adeno-associated virus (SCAAV) as a helper virus vector to load TVA receptor and rabies virus outer membrane glycoprotein (RVG) respectively, and packaged into virus for specific recognition andretrograde transsynaptic marker of ENVA outer membrane wrapped defective recombinant rabies virus. As shown by that live test result, the recombinant defective rabies virus RV-[delta]G-X-ENVA and helper virus SCAAV combined system, can realize fast retrograde transsynaptic labeling, which saves 1-2 week experiment time, saves material resources and manpower, provides a better research tool for theapplication of defective rabies virus in neural network reverse transsynaptic marker, and lays a good technical support for the structure and function analysis of neural network.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method and application of rapid retrograde transsynaptic labeling of nerve cells. Background technique [0002] Analyzing the neural network connections of the brain is the basis for understanding the working mechanism of the brain and the network variation mechanism of brain diseases. Traditional neural network tracing methods, a small number of protein tracers, such as WGA, HRP, etc., can initially realize the projection relationship between brain nuclei, but these tracing methods have signal indirection, non-specific direction, trans-synaptic signal Serious attenuation and other issues. The above-mentioned traditional tracing methods have promoted people's understanding of the structure of the brain's neural network, but it is difficult to study the complex neural network formed by multiple brain regions and various types of neurons through synaptic connections. Tr...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N15/66A61K49/00
CPCA61K49/0047A61K49/0097C12N15/66C12N15/86C12N2750/14143
Inventor 林坤章苏鹏何晓斌徐富强
Owner WUHAN INST OF PHYSICS & MATHEMATICS CHINESE ACADEMY OF SCI
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