CN111808862A - GABA (gamma-aminobutyric acid) energy neuron specific promoter and application thereof - Google Patents

Abstract

The invention discloses a GABAergic neuron specific promoter and application thereof, wherein the promoter is a brain neuron specific promoter obtained from a genome of a C57BL/6J mouse, and can regulate and control the specific expression of genes in GABAergic neurons. The promoter is loaded into an adeno-associated virus vector and is infected in a mouse brain, and a target gene can be mediated to be specifically expressed in GABAergic neurons, so that GABAergic nerve groups existing in the brain are marked or controlled. The promoter has wide application value and market prospect in the aspects of structural and functional analysis of GABAergic neuron groups in specific brain regions, disease model establishment, gene therapy and the like.

Description

GABA (gamma-aminobutyric acid) energy neuron specific promoter and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a promoter for regulating and controlling specific expression of genes in aminobutyric acid (GABA) neurons.

Background

Analyzing the brain neural network connection is the basis for understanding the brain working mechanism and brain disease network mutation mechanism. The brain network is composed of a variety of nerve cells, which mainly include two major classes of neurons and glial cells (neuroglia), each playing a different role in the brain. Neuronal cells are highly differentiated cells, which constitute the basic structural and functional units of the nervous system, have the functions of sensing stimulation and conducting excitation, and are classified into aminobutyric acid (GABA) neurons, glutamatergic neurons, dopaminergic neurons, cholinergic neurons, serotonin neurons, and the like, according to the produced neurotransmitters. In the nervous system, the number of glial cells is several tens of times greater than that of neurons, and these are generally classified into astrocytes, microglia, oligodendrocytes, and schwann cells in the peripheral nervous system. With the development of neuroscience, different types of nerve cells are found to have different network structure connection and functions, and the variation of the nerve cells can cause different brain diseases and behavior disorders. Therefore, it is necessary to label and manipulate specific types of nerve cells in specific brain regions in order to analyze their structures and functions, and to provide theoretical basis and technical support for the treatment of brain diseases.

Currently, researchers rely primarily on transgenic animals for the resolution of specific types of neurons. However, transgenic animals, such as mice, rats, dogs, pigs, monkeys, etc., have long breeding cycles, high feeding costs, and are not able to flexibly carry target genes for nerve cell manipulation and disease treatment; therefore, it is of great interest to continue to develop methods that can be used to label and manipulate specific types of neurons.

With the development of bioinformatics and molecular biology, the use of specific promoters to mediate exogenous gene expression has become a highly effective approach for specific tissue targeting and manipulation and gene therapy. The invention provides a specific promoter which can mediate the expression of a target gene in GABAergic neurons specifically so as to mark or control GABAergic neuron groups existing in the brain. The promoter has wide application value and market prospect in the fields of neuroscience, brain disease model establishment, gene therapy and the like.

Disclosure of Invention

One of the purposes of the invention is to provide a brain neuron specific promoter VGAT obtained from the genome of a mouse of a C57BL/6J strain, and the nucleotide sequence of the promoter is shown as SEQ ID NO. 1.

Viral vectors containing the promoter VGAT are also within the scope of the invention. The virus vector comprises an adeno-associated virus vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a herpes simplex virus amplicon vector, a pseudorabies virus vector, a baculovirus vector, a poxvirus vector and the like.

The promoter VGAT can regulate the specific expression of a target gene in aminobutyric acid (GABA) energy neurons. In a specific example of the present invention, the promoter, VGAT, was loaded into an adeno-associated viral vector and infected in a Zona Incerta (ZI) region of mouse brain, and the result showed that the VGAT promoter was able to specifically initiate the expression of EYFP gene in GABA-aminobutyric acid (GABA) -competent neurons.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. provides a GABAergic neuron specific promoter which can mediate the expression of a target gene in GABAergic neurons specifically;

2. compared with the traditional transgenic method, the specific promoter mediated target gene expression is more flexible, more convenient to apply and lower in cost;

3. specific promoters can be loaded into a desired vector while therapeutic genes can be expressed for gene therapy, which cannot be achieved with transgenic animals.

Drawings

FIG. 1 is a map of a recombinant expression vector.

FIG. 2 is a test of VGAT promoter in vivo; wherein, the graph A shows that DAPI stains cell nucleus; panel B shows green fluorescence signals for VGAT-initiated expression; panel C is the in situ hybridization signal for gabaergic neurons; panel D shows the effect of co-targeting between fluorescent signals of VGAT promoter-mediated expression and GABAergic neurons. The results indicate that the VGAT promoter can specifically initiate the expression of the target gene in GABA (GABA) ergic neurons.

Detailed Description

Example 1: isolation and characterization of promoter VGAT

1. Primer design

According to the whole genome sequence of the mouse C57BL/6J variety provided in NCBI, an amplification primer is designed according to the sequence of mouse VGAT gene, and according to the characteristics of the selected carrier and target gene, a proper enzyme cutting site is added in front of the primer. The primer sequences are as follows:

an upstream primer: 5' -CGCACGCGTCACCGACCTCCCAGGGGTGC-3’；

A downstream primer: 5' -CCTACCGGTTCCCTAGCTCAGCTTTCTCC-3’。

Wherein the upstream primer carries MluI restriction site (ACGCGT), and the downstream primer carries AgeI restriction site (ACCGGT), which are underlined respectively. The primers were synthesized by Biotechnology engineering (Shanghai) Inc.

2. Promoter VGAT cloning

The VGAT promoter fragment is PCR amplified by using the genome DNA of a mouse C57BL/6J variety as a template and using upstream and downstream primers, wherein the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 59 ℃ for 30s, extension at 72 ℃ for 1.5min, and 30 cycles; extension at 72 ℃ for 10 min. After the PCR amplification product is detected and confirmed by 1% agarose gel electrophoresis, the target fragment is recovered by cutting the gel and sent to the company Limited in Biotechnology engineering (Shanghai) for sequencing verification, and the result shows that: the amplified sequence is a VGAT promoter sequence, and the nucleotide sequence is shown in SEQ ID NO. 1.

Example 2: functional validation of promoter VGAT

Construction of recombinant expression vector

The VGAT promoter verified by sequencing is subjected to double enzyme digestion by MluI and AgeI and then gel running and recovery, T4 ligase (purchased from TaKaRa company) is connected into an adeno-associated virus vector pAAV-CamkIIa-EYFP-WPRE-hGH polyA (purchased from Wuhan Shuzo dense brain science and technology Co., Ltd.) which is also subjected to double enzyme digestion by MluI and AgeI, a connecting product is transformed into an escherichia coli StbI3 competent cell, the escherichia coli StbI competent cell is placed in a 35-degree culture box for overnight, a single clone is selected for colony PCR identification, the positive colony is subjected to amplification culture and plasmid extraction, the plasmid is verified and sequenced by double enzyme digestion by MluI and AgeI, and the plasmid with correct sequencing is named pAAV-VGAT-EYFP-WPRE-hGH A. The map of the constructed expression vector is shown in FIG. 1.

Second, viral preparation of recombinant expression vectors

By using conventionalThe three-plasmid package adeno-associated virus method comprises the steps of carrying out virus package on recombinant expression vectors, concentrating and purifying by using an iodixanol gradient centrifugation method, and finally detecting the titer of the recombinant adeno-associated virus by using a SYBR Green qPCR method to finally obtain the titer of the rAAV-VGAT-EYFP-WPRE-hGH polyA virus of 5 multiplied by 10¹²vg/ml。

Three, in vivo testing of VGAT promoter

rAAV-VGAT-EYFP-WPRE-PA virus is injected into a zone of Zona Incerta (ZI) of a 3-month-old C57BL/6J mouse (purchased from Hunan Slek Jingshoda laboratory animals Co., Ltd.) through brain stereotactic mode, after the virus is sufficiently expressed for 4 weeks, 0.5ml of 1% sodium pentobarbital-0.9% sodium chloride Solution is injected into the abdominal cavity of the mouse for anesthesia, and Phosphate Buffer Solution (PBS) treated by Diethyl Pyrocarbonate (DEPC) and 4% Paraformaldehyde Solution (PFA) are used for heart perfusion to strip brain tissue. Mouse brain tissue was fixed with DEPC-treated PFA solution for 4 hours, then dehydrated with DEPC-treated 30% sucrose-PBS solution, and the dehydrated brain tissue was fully embedded with a tissue embedding medium and cut into brain slices of 50 μm thickness with a cryomicrotome. mRNA of a neuron of gamma-aminobutyric acid (GABA) In a ZI region was labeled In red by an In Situ Hybridization technique (ISH) using Vgat as a probe (gene sequence derived from ALLEN BRAIN Slc32a1, http:// mouse. blue-map. org/gene/show/22105), and then eYFP fluorescent protein expressed In the neuron by AAV (eYFP fluorescence is quenched during ISH) was counterstained In green by immunohistochemical staining using anti-GFP (purchased from Abcam, Cat. RTM. ab290) antibody. And finally, preparing a brain tissue sample containing the ZI region into a slice, and imaging the slice by using a laser scanning confocal microscope, wherein the wavelengths of exciting light are 488nm and 594nm respectively. The in vivo detection result is shown in FIG. 2, and most of the green fluorescence signals and the red fluorescence signals are labeled together, which indicates that the VGAT promoter can specifically initiate the expression of the target gene in gamma-aminobutyric acid (GABA) neurons.

Sequence listing

<110> institute of precision measurement, science and technology innovation, of the Chinese academy of sciences

<120> GABA energy neuron specific promoter and application thereof

<160>1

<170>SIPOSequenceListing 1.0

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<211>1300

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

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caccgacctc ccaggggtgc acctctccag cctcggtcgc gcttccgaaa cctttggtgc 60

ccccttttcc tggtcccgac cccccacctc acgccccctg gtctggacag catctccccc 120

tcgccgccct ccgcccacgc accgcctgac tccgaggggt gcgagcgcat tgggctgcgc 180

ccgcgtgggg gcgccgcgcc agcctcgcgt agctgttctg acgctgccgt cgccgccgcc 240

ctccgcagcc cagccggcac ccgcaccagc tctgcagtgc actcgtcgcc tctcgggccg 300

gtcccaccaa gagccagact gtcgtgaccg gggccagcct cgaacgtcag gcgcgagggt 360

catgagccag agcgccctgg ggcgccgcgc ggagacccag cggagatagc agtcctcgct 420

gccttgacgc gcgcccgccg cgtccccaga cccttctgtc cttttctccc gccccgccgc 480

cgccatggcc accctgctcc gcagcaagct gaccaatgtg gccacctccg tgtccaacaa 540

gtcccaggcc aaggtgagcg gcatgtttgc caggatgggg tttcaggcgg ccaccgatga 600

ggaagcggtg ggcttcgcgc actgcgacga tctcgacttt gagcatcgcc agggcctgca 660

gatggacatc ctgaaatcgg aaggcgagcc ctgcggagac gagggcgcag aagctcccgt 720

cgagggagac attcattatc agcgcggcgg cgctcctctg ccaccctctg gctccaagga 780

ccaggccgtg ggagctggtg gggagttcgg gggtcacgac aaacccaaga tcacggcgtg 840

ggaagcgggc tggaacgtga caaatgccat tcaggtgagt gcgggatccc caaatctgct 900

tgccatcacc ccccacctga gctgtccttg ccaggctctg cccccacacg aaccccgcag 960

aggtctaggt ttcaatgccg ccttctccca ggactggata atttatctcc ccttctctga 1020

ccttcctagc catagggatc tacgccccca ggcggtgttc tcctcagatc cactcttgct 1080

gttcctgcta caggacagcc tggctgaggt ttggggtggg ggcgggggcg ggtcacaaat 1140

ccaaagaccc tacaaaagct ggagacatgg ggaagggcgg gagtcggggt gggggaagaa 1200

tgaagaagaa agaaaatcaa gatctggaga gttagccttg agctccagaa tggagtccca 1260

gctgcatttt cggaggacag ggagaaagct gagctaggga 1300

Claims (3)

1. The promoter is characterized in that the nucleotide sequence of the promoter is shown as SEQ ID NO. 1.

2. A viral vector comprising the promoter of claim 1.

3. Use of the promoter of claim 1 or the viral vector of claim 2 for regulating neuronal specific expression of a gene of interest.

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