A method and application of rapid retrograde transsynaptic labeling of nerve cells

A neuronal and fast technology, applied in the biological field, can solve the problems of limited double-stranded AAV load, long experimental period, and large auxiliary gene RVG size, etc.

Active Publication Date: 2022-04-01
WUHAN INST OF PHYSICS & MATHEMATICS CHINESE ACADEMY OF SCI
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  • Claims
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AI Technical Summary

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

Method used

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  • A method and application of rapid retrograde transsynaptic labeling of nerve cells
  • A method and application of rapid retrograde transsynaptic labeling of nerve cells
  • A method and application of rapid retrograde transsynaptic labeling of nerve cells

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Embodiment 1

[0033] A method for rapid retrograde trans-synaptic labeling of nerve cells, comprising the following steps:

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

[0035] 1. Construction of Helper Virus Vectors

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

[0037]SCAAV-hSyn-DIO-EYFP was double-cut with NheI / A...

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Abstract

The invention belongs to the field of construction of helper virus, and discloses a method and application of rapidly retrograde transsynaptic labeling of nerve cells. The present 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 package them into viruses for the specificity of defective recombinant rabies virus wrapped in ENVA outer membrane. Sex recognition and retrograde transsynaptic labeling. The results of in vivo tests show that the combined system of recombinant defective rabies virus RV-△G-X-ENVA and the helper virus SCAAV established by the present invention can realize rapid retrograde trans-synaptic labeling, saving 1.5 lb compared with the traditional single-chain AAV virus helper method. ‑2 weeks of experiment time, saving material and manpower, providing a better research tool for the application of defective rabies virus in neural network reverse transsynaptic labeling, and laying a good technology for the structure and function analysis of neural network support.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method and application of rapid retrograde trans-synaptic labeling of nerve cells. Background technique [0002] Analyzing the neural network connections in 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 few protein tracers, such as WGA, HRP, etc., can initially realize the projection relationship between brain nuclei, but these tracing methods have indirect signal, direction non-specific, trans-postsynaptic signal. Serious attenuation and so on. The above-mentioned traditional tracing methods have promoted people's understanding of the structure of the neural network in the brain, but it is difficult to be used 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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Patent Type & AuthorityPatents(China)
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
OwnerWUHAN INST OF PHYSICS & MATHEMATICS CHINESE ACADEMY OF SCI