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GABAergic neuron specific promoter and application thereof

A technology capable of neurons and promoters, applied in the field of genetic engineering, can solve the problems of long breeding cycle, high feeding cost, inability to flexibly carry target genes, etc., and achieve the effect of low cost and convenient application.

Pending Publication Date: 2020-10-23
INNOVATION ACAD FOR PRECISION MEASUREMENT SCI & TECH CAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, transgenic animals, such as mice, rats, dogs, pigs, and monkeys, have long reproductive cycles, high feeding costs, and cannot flexibly carry target genes for manipulation of nerve cells and treatment of diseases; methods for labeling and manipulating specific types of neurons are of great importance

Method used

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  • GABAergic neuron specific promoter and application thereof
  • GABAergic neuron specific promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1: Isolation and Identification of Promoter VGAT

[0015] 1. Primer design

[0016] According to the complete genome sequence of the mouse C57BL / 6J variety provided by NCBI, the amplification primers were designed according to the sequence of the mouse VGAT gene, and appropriate restriction sites were added in front of the primers according to the characteristics of the selected vector and target gene. The primer sequences are as follows:

[0017] Upstream primer: 5'-CGC ACGCGT CACCGACCTCCCAGGGGTGC-3';

[0018] Downstream primer: 5'-CCT ACCGGT TCCCTAGCTCAGCTTTCTCC-3'.

[0019] Wherein, the upstream primer has a MluI restriction site (ACGCGT), and the downstream primer has an Agel restriction site (ACCGGT), which are respectively underlined. Primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0020] 2. Promoter VGAT clone

[0021] Using the genomic DNA of the mouse C57BL / 6J strain as a template, the VGAT promoter fragment was amplifi...

Embodiment 2

[0022] Example 2: Functional verification of the promoter VGAT

[0023] 1. Construction of recombinant expression vector

[0024] The sequence-verified VGAT promoter was digested with MluI and AgeI and recovered by running the gel, and connected with T4 ligase (purchased from TaKaRa Company) into the adeno-associated virus vector pAAV-CamkIIa-EYFP also double-digested with MluI and AgeI -WPRE-hGH polyA (purchased from Wuhan Privy Brain Science and Technology Co., Ltd.), the ligation product was transformed into Escherichia coli StbI3 competent cells, placed in a 35-degree incubator overnight, single clones were picked for colony PCR identification, and positive colonies were expanded for culture and plasmid extraction, the plasmid was verified and sequenced by MluI and AgeI double enzyme digestion, and the sequenced correct plasmid was named pAAV-VGAT-EYFP-WPRE-hGH polyA. The map of the constructed expression vector is as follows figure 1 shown.

[0025] 2. Virus preparatio...

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Abstract

The present invention discloses a GABAergic neuron-specific promoter and an application thereof. The promoter is a brain neuron-specific promoter obtained from a mouse genome of a C57BL / 6J breed and can regulate specific expression of genes in GABAergic neurons. The promoter is loaded into an adeno-associated virus vector, infects a mouse brain in vivo and can mediate the specific expression of the target gene in the GABAergic neuron, thereby marking or manipulating GABAergic neuron groups existing in the brain. The promoter has a wide range of application value and market prospects in structure and function analysis of the GABAergic neuron groups in specific brain regions, disease model establishment, gene therapy, etc.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a promoter for regulating the specific expression of genes in aminobutyric acid (GABA) neurons. 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. The brain network is composed of various nerve cells. Nerve cells mainly include neurons and glial cells (neuroglia), which play different roles in the brain. Neuron cells are highly differentiated cells that constitute the basic structural and functional units of the nervous system. They have the functions of feeling stimuli and conducting excitement. Aminergic neurons, dopaminergic neurons, cholinergic neurons, and serotonergic neurons. In the nervous system, the number of glial cells is dozens of times more than that of neurons, and can be generally divided into astro...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/864
CPCC07K14/47C12N15/86C12N2750/14143
Inventor 林坤章徐富强文鹏杰何晓斌
Owner INNOVATION ACAD FOR PRECISION MEASUREMENT SCI & TECH CAS
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