CN114231565A - rAAV capable of being used for in vivo detection of cell type specific nerve connection and application thereof - Google Patents
rAAV capable of being used for in vivo detection of cell type specific nerve connection and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant adeno-associated virus for in vivo detection of cell type specific nerve connection and application thereof in nerve structure loop tracing. AAV2-retro serotype recombinant adeno-associated virus rAAV2-retro-CAG-DIO-AQP1-2A-EGFP-WPRE-pA carrying fusion gene of cre dependent expression type 1 aquaporin and green fluorescent protein is injected into the brain of cre strain transgenic mice, virus infects neurons and expresses AQP1 protein in mediated neurons, the living brain is observed by a magnetic resonance imaging DWI method, cell type specific nerve connection between other brain areas and the infected neurons at the injection site is displayed in the living brain, the cell type, direction and connection progression of nerve connection can be definitely obtained, and a solid foundation is provided for functional analysis of nerve projection.
Description
Technical Field
The invention belongs to the technical field of virus vectors, and particularly relates to a recombinant adeno-associated virus carrying cre-dependent expressed type 1 aquaporin and green fluorescent protein fusion gene, and application thereof in neural structure loop tracing and function analysis.
Background
The brain is a complex and sophisticated system that is the core of information processing control for individuals. The human brain contains hundreds of billions of neurons, which contain hundreds of cell types, and the neurons form a huge neural network through synaptic connections, which forms the structural basis for the brain to process information and regulate and control behaviors. The key to understanding the neural mechanism of brain work is to analyze the roles played by the subpopulations of neurons of different cell types in the neural network, and to reveal the number, location, morphology, input/output connections and corresponding physiological functions of specific types of neurons in specific brain regions.
The nerve network which infects neurons and marks the brain by using the neurotropic tool virus carrying the reporter gene is the commonly used technology for tracing the structural nerve network at present. The tool virus vector can mediate the exogenous gene to express stably and efficiently in various cells, and can limit the expression of the exogenous gene in specific cells through molecular elements, so that the tool virus vector can be used for marking cell type specific neural networks and is beneficial to finely analyzing the neural networks.
Recombinant Adeno-Associated Virus (rAAV) is a viral vector commonly used for neural circuit tracing, and has a plurality of capsid proteins corresponding to a plurality of serotypes, and AAV infection characteristics of different serotypes are different. The rAAV2-retro (retro for short) serotype AAV can enter neurons through axon terminals, reversely transport to neuron cell bodies and transduce exogenous genes, and can be used for marking the direct projection of nerve fibers to an upstream network of a specific brain region. Compared with other virus vectors for marking upstream nerve connection, the rAAV2-retro has the advantages of low toxicity, stable and durable foreign gene expression, high biological safety (compatible with living body MRI scanning) and the like. By combining with Cre-loxP system, the exogenous gene carried by AAV can be specifically expressed in specific type of neurons, which is beneficial to the research of cell type specific neural network. For example, DIO elements can be inserted into rAAV genomes such that expression of the foreign gene is controlled by cre recombinase. The DIO element is Double-floxed invader organization, which consists of two different LoxP sites, and under the condition that Cre recombinase is not available, the target gene is not expressed, and under the condition that Cre exists, the target gene is overturned to the correct direction, so that the target gene can be expressed.
However, the existing neurotropic viruses often carry fluorescent protein genes, and the obtained labeling results are mostly shown by in vitro fluorescent images, so that the analysis of the whole brain structure neural network at the living body level is difficult to realize. The development of efficient in vivo detection methods for neural networks in whole brain structure remains a great challenge in neural network research, and the development of methods for detecting cell type-specific neural junctions at the in vivo level is particularly difficult.
Magnetic Resonance Imaging (MRI) is an imaging method combining many advantages of non-invasive, non-ionizing radiation, high-penetrability, and suitability for soft tissue, and has been widely used in clinical diagnosis and scientific research. Genes encoding proteins that can be detected by MRI are called MRI reporter genes. MRI reporter genes are currently used to monitor processes such as gene expression and cell migration in living animals. The aquaporin 1(AQP1) gene is a novel MRI reporter gene reported in recent years. Its expression product, Aquaporin (AQP), is a group of transmembrane transporters with high permselectivity for water. The over-expression of AQP1 protein can affect the Diffusion rate of water molecules across cell membranes, generate obvious change of Diffusion Weighted Imaging (DWI) signals in cells and living tissues, present dark signals in DWI-MRI images and present high signals in Apparent Diffusion Coefficient (ADC) maps. Compared with other MRI reporter genes, AQP1 has the advantages of no dependence on metal ions, high sensitivity, no obvious cytotoxicity and the like.
In conclusion, the rAAV2-retro vector carries the MRI reporter gene AQP1, so that the MRI-based nerve junction living body detection can be realized, and the rAAV 2-retro-mediated AQP1 expression can be targeted to neurons of a specific cell type by combining with a Cre-loxP system, so that the nerve junction of the specific cell type can be detected at the living body level.
Disclosure of Invention
The invention aims to provide a recombinant adeno-associated virus vector which can be used for detecting cell type-specific nerve connection in vivo.
Another objective of the invention is to provide the application of the recombinant adeno-associated virus vector in neural structure loop tracing, wherein the recombinant adeno-associated virus vector can be used for in vivo detection of cell type-specific neural connection.
In order to achieve the purpose, the invention adopts the following technical scheme:
recombinant adeno-associated virus core plasmid: the core plasmid carries expression frames of cre-dependent expressed aquaporin AQP1 gene and fluorescent protein reporter gene. Specifically, the plasmid is characterized in that a CAG promoter (shown in SEQ ID NO. 1), a DIO element (shown in SEQ ID NO. 2), an AQP1 gene (shown in SEQ ID NO. 3), a 2A connection sequence (shown in SEQ ID NO. 4), an EGFP gene (shown in SEQ ID NO. 5), a WPRE post-transcriptional regulation element (shown in SEQ ID NO. 6) and a polyA sequence (shown in SEQ ID NO. 7) are sequentially inserted into the middle of an ITR of a rAAV vector plasmid to construct a recombinant adeno-associated virus core plasmid pAAV-CAG-DIO-AQP 1-2A-EGFP-WPRE-pA.
Recombinant adeno-associated virus: the virus takes pAAV-CAG-DIO-AQP1-2A-EGFP-WPRE-pA as a core plasmid to prepare the recombinant adeno-associated virus, and the specific method comprises the following steps: co-transfecting an HEK293T cell with an auxiliary plasmid, a rAAV2-retro (retro for short) serotype plasmid and a core plasmid required by packaging rAAV, collecting the centrifuged cell precipitate, and purifying the recombinant adeno-associated virus rAAV2-retro-CAG-DIO-AQP 1-2A-EGFP-WPRE-pA.
The application of the recombinant adeno-associated virus in detecting cell type specific nerve connection at living body level comprises the following steps: the recombinant adeno-associated virus is injected into the brain of a cre transgenic mouse, the virus infects neurons and mediates AQP1 protein expression in the neurons, the living brain is observed by a magnetic resonance imaging (DWI) method, cell type-specific nerve connection between other brain areas and the infected neurons at the injection site is displayed in the living brain, and the specific cell type is determined by the cre strain of the transgenic mouse. In a specific embodiment of the invention, the recombinant adeno-associated virus rAAV2-retro-CAG-DIO-AQP1-2A-EGFP-WPRE-pA is injected into the brain area A of the Thy1-cre transgenic mouse, and after a period of time, DWI-MRI imaging shows that DWI signal change caused by AQP1 exists in the brain area B, so that nerve connection exists between the brain area A and the brain area B, the nerve connection is that Thy1 positive specific neurons in the brain area B have direct axon projection to the brain area A, and the cell type, direction and connection progression of the nerve connection are determined. The unambiguous neural structure connection information thus obtained provides a structural basis for the functional analysis of the neural projection from the subsequent brain B region to the brain a region.
Compared with the prior art, the method has the following advantages:
1. the rAAV2-retro serotype recombinant adeno-associated virus adopted in the invention can enter neurons from axon terminals and reversely enter neuron cell bodies through axon transportation, and can definitely indicate nerve connection;
2. the AQP1 protein mediated and expressed by the recombinant adeno-associated virus rAAV2-retro-CAG-DIO-AQP1-2A-EGFP-WPRE-pA is a DWI-MRI contrast agent, and compared with a common MRI report protein ferritin, the DWI-MRI contrast agent has high sensitivity and can generate an MRI contrast effect more quickly without depending on metal ions;
3. the method provided by the invention can be used for observing the brain nerve connection at the living body level, is suitable for long-term observation, self-contrast experiments before and after treatment and the like, and has important significance for the study of the brain nerve structure network;
4. the neural network structure connection obtained by the method is cell type specific neural connection, the specific cell type is determined by the transgenic mouse cre strain, and the method can be flexibly applied to detecting the cell type specific neural connection;
5. the neural network structure obtained by the method provided by the invention has definite connection direction, namely the neural network structure is a reverse mark;
6. the neural network structure obtained by the method provided by the invention has definite connection progression, namely direct connection instead of multi-level synaptic connection.
Drawings
FIG. 1 is a schematic diagram of the genome of rAAV-CAG-DIO-AQP1-2A-EGFP-WPRE-pA virus. An exogenous expression frame is arranged between ITRs on two sides, CAG is a promoter, AQP1 is a aquaporin gene, 2A is a connecting sequence, EGFP is a green fluorescent protein gene, WPRE is a post-transcriptional enhancement element, and pA is a polyA sequence. Black and white triangles represent different LoxP sites, respectively. In the presence of Cre recombinase, the AQP1-2A-EGFP sequence in the middle of the LoxP site is inverted to the normal expression direction.
FIG. 2 shows the MRI and fluorescence imaging results of mouse brains injected with rAAV2-retro-CAG-DIO-AQP 1-2A-EGFP-WPRE-pA. A is the difference result of the ADC values calculated from the DWI-MRI image, and the brain regions Ctx, CPU, Ins, Tha and BLA indicated by arrows are the brain regions with significantly increased ADC values (decreased DWI signals); and B is the green fluorescence imaging result of the mouse brain slice. Green is the fluorescent signal of EGFP expressed by virus mediation, blue is the DAPI staining signal of nucleus.
FIG. 3 shows the Cre immunohistochemical detection result of mouse brain tablet injected with rAAV2-retro-CAG-DIO-AQP 1-2A-EGFP-WPRE-pA. The red fluorescent signal is an immunohistochemical staining signal of Cre, red and green fluorescent signals are respectively arranged in Ctx, Tha and BLA brain regions, and the EGFP signal and the Cre immunohistochemical signal are greatly overlapped.
Detailed Description
The technical solutions of the present invention, if not specifically mentioned, are conventional in the art, and the reagents and materials, if not specifically mentioned, are commercially available. The specific embodiments illustrated herein are merely illustrative of the invention and are not intended to be limiting.
Example 1: construction of plasmid pAAV-CAG-DIO-AQP1-2A-EGFP-WPRE-pA carrying cre dependent expression AQP1 and EGFP fusion gene and preparation of recombinant adeno-associated virus rAAV2-retro-CAG-DIO-AQP1-2A-EGFP-WPRE-pA
The DIO element in pAAV-CAG-DIO-AQP1-2A-EGFP-WPRE-pA enables the target gene to start to be expressed only in the presence of Cre. In order to improve the expression level of a target gene, a eukaryotic strong promoter CAG promoter is selected. To express AQP1 gene fused with green fluorescent protein (EGFP) reporter gene, we selected the 2A self-splicing polypeptide coding sequence as the junction sequence of AQP1 gene and EGFP gene. In order to improve the stability of the target gene after transcription, WPRE (human polyporus frondosus) posttranscriptional regulatory elements and polyA sequences are added at the 3' end of the target gene. Inserting a CAG promoter (shown in SEQ ID NO. 1), a DIO element (shown in SEQ ID NO. 2), an AQP1 gene (shown in SEQ ID NO. 3), a 2A connecting sequence (shown in SEQ ID NO. 4), an EGFP gene (shown in SEQ ID NO. 5), a WPRE post-transcriptional regulatory element (shown in SEQ ID NO. 6) and a polyA sequence (shown in SEQ ID NO. 7) in the middle of an AAV terminal repetitive sequence (ITR) in a core plasmid pAAV-MCS for packaging rAAV by using a conventional molecular cloning method; the plasmid pAAV-CAG-DIO-AQP1-2A-EGFP-WPRE-pA containing the genome of the desired rAAV was obtained. The genome schematic diagram of the prepared rAAV2-retro-CAG-DIO-AQP1-2A-EGFP-WPRE-pA is shown in figure 1.
The packaging and purification steps of the recombinant adeno-associated virus rAAV2-retro-CAG-DIO-AQP1-2A-EGFP-WPRE-pA are as follows:
HEK293T cells were first plated into 10cm cell dishes and cultured to 80% density, and three plasmids required for packaging rAAV (helper plasmid pAAV-helper, serotype plasmid pAAV-RC2-retro, core plasmid pAAV-CAG-DIO-AQP1-2A-EGFP-WPRE-pA) were co-transfected into HEK293T cells at a molar ratio (1:1: 1). After transfection for 72h, the adherent cells producing the virus were scraped off by cell scraping and centrifuged, and the centrifuged cell pellet was collected. Then, cell pellet lysate is treated with nuclease, and rAAV in the virus supernatant is concentrated with PEG/NaCl pellets. And (3) separating and purifying the lysate supernatant containing the rAAV by iodixanol density gradient ultracentrifugation, subpackaging the purified rAAV, and freezing and storing in a refrigerator at the temperature of-80 ℃. The titer of rAAV virus was determined by real-time fluorescent quantitative PCR using a CFX Connect fluorescent quantitative PCR instrument from BioRad.
Example 2: MRI imaging and fluorescence imaging observation of mouse brain injected with rAAV2-retro-CAG-DIO-AQP1-2A-EGFP-WPRE-pA
rAAV2-retro-CAG-DIO-AQP1-2A-EGFP-WPRE-pA virus is injected into dorsal striatum brain area (CPu, coordinate: AP-0.5 mm; ML-2 mm; DV-3.3mm) of Thy1-cre mouse through brain stereotaxic injection, and the titer is 5 multiplied by 1012vg/ml, injection volume 1. mu.L. The control virus rAAV2-retro-CAG-DIO-EGFP-WPRE-pA is injected into the dorsal striatal brain area of a control group Thy1-cre mouse, and the titer of the control virus rAAV2-retro-CAG-DIO-EGFP-WPRE-pA is 5 multiplied by 1012vg/ml, NoteThe shot volume was 1 μ L.
After 3 weeks, the animal brains were subjected to in vivo MRI observations using a 7.0T magnetic resonance imager, wherein the diffusion-weighted imaging sequence used was a STEAM-based SE-DWI sequence with the parameters: TR is 3000ms, TE is 24ms, the average number of times is 2, and the total sequence duration is 51min12 s. The diffusion gradient duration is 7ms, the diffusion gradient interval time is 100ms, and the b value is 1000s mm-2. FOV of 1.8X 1.8cm2The spatial resolution is 0.18X 0.18mm and the layer thickness is 0.8 mm. From the DWI images of the experimental and control groups, the ADC maps of the two groups were calculated and the differences in ADC values were compared. FIG. 2A shows that the ADC values of the brain regions Ctx, CPU, Ins, Tha and BLA of the experimental group are significantly increased relative to the control group. This indicates that the virus successfully infected neurons, transported retrogradely from the injection site CPU via axons to multiple upstream brain regions (Ctx, Ins, Tha and BLA), and mediated the expression of AQP1 protein that altered the DWI-MRI signal. Thus indicating that this method can detect at the living level the linkage of Thy1 positive neurons of the upstream brain regions (Ctx, Ins, Tha and BLA) to the nerves of the CPU.
After the MRI experiment, mice were anesthetized, and after cardiac perfusion, mouse brains were removed and cryo-sectioned. Mouse brain sections were then DAPI stained to reveal nuclear locations. Fluorescence imaging was then performed, and the results are shown in fig. 2B, green for the fluorescent signal of EGFP expressed by virus and blue for the DAPI staining signal of the nucleus. The EGFP high expression region DWI-MRI signal change regions displayed by the green fluorescence signals are consistent in height and are all brain regions such as Ctx, CPU, Ins, Tha and BLA, and the in-vivo detection result of MRI is verified by the nerve connection tracing result of in-vitro fluorescence imaging. It was also demonstrated that DWI-MRI signal changes were caused by high expression of virus-borne AQP 1.
Example 3: cre immunohistochemical detection of mouse brain tablet injected with rAAV2-retro-CAG-DIO-AQP1-2A-EGFP-WPRE-pA
rAAV2-retro-CAG-DIO-AQP1-2A-EGFP-WPRE-pA virus is injected into dorsal striatum brain area (CPu, coordinate: AP-0.5 mm; ML-2 mm; DV-3.3mm) of Thy1-cre mouse through brain stereotaxic injection, and the titer is 5 multiplied by 1012vg/ml, injection volume 1. mu.L. After 3 weeks, the animals' brains were advanced using a 7.0T magnetic resonance imagerAnd performing living body DWI-MRI observation. After the MRI experiment, mice were anesthetized, and after cardiac perfusion, mouse brains were removed and cryo-sectioned. Mouse brain sections were then immunohistochemically stained with rabbit-derived Cre antibody and goat anti-rabbit secondary antibody (red) to reveal the localization of Cre protein. The neuron expressing Cre protein in Thy1-Cre mouse is positive Thy1 neuron. In fig. 3 the red fluorescence signal is the immunohistochemical staining signal for Cre, red and green fluorescence signals are present in all of the Ctx, Tha and BLA brain regions, and the EGFP signal largely overlaps with the Cre immunohistochemical signal, indicating overlap of EGFP with Cre protein expression. Therefore, the neurons marked by green fluorescent protein in Ctx, Tha and BLA of the upstream brain region are proved to be positive by Thy1, namely the neurons marked by rAAV 2-retroro-CAG-DIO-AQP 1-2A-EGFP-WPRE-pA virus and connected to the upstream of the CPU detected by MRI living body are positive by Thy 1.
Sequence listing
<110> institute of precision measurement, science and technology innovation, of the Chinese academy of sciences
<120> rAAV capable of being used for in vivo detection of cell type specific nerve connection and application thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1460
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 60
tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 120
gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 180
acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 240
accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 300
gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca 360
attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg 420
gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag gcggagaggt 480
gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg 540
cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg ggagtcgctg cgacgctgcc 600
ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg 660
cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct 720
tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg 780
agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga 840
gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc 900
tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg 960
gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag 1020
ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg 1080
agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc 1140
cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg 1200
agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga 1260
gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc 1320
aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg 1380
cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc 1440
gccgccgtcc ccttctccct 1460
<210> 2
<211> 130
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ataacttcgt ataaagtatc ctatacgaag ttatatcaaa ataggaagac caatgcttca 60
ccatcgaccc gaattgccaa gcatcaccat cgacacataa cttcgtataa tgtatgctat 120
acgaagttat 130
<210> 3
<211> 807
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atggccagcg agttcaagaa gaagctgttc tggagggccg tggtggctga gttcctggcc 60
accaccctgt tcgtgttcat cagcatcggc agcgccctgg gcttcaagta ccctgtgggc 120
aacaaccaga cagccgtgca agacaacgtg aaggtgtccc tggccttcgg cctgagcatc 180
gccaccctgg cccagtcggt gggacacatc tccggcgccc acctgaaccc tgccgtgacc 240
ctgggcctgc tgctgagctg tcagatcagc atctttagag ccctgatgta catcatcgcc 300
cagtgtgtgg gtgccatcgt ggccaccgcc atcctgagcg gcatcaccag cagcctgacc 360
ggcaacagcc tgggcagaaa cgacctggcc gacggcgtga acagcggcca gggcctgggc 420
atcgagatca tcggcacact gcagctggtg ctgtgcgtgc tggctacaac cgacaggagg 480
agaagagacc tgggaggatc cgcccctctg gctattggcc tgagcgtggc cctgggacat 540
ctgctggcta ttgattatac aggatgtggc attaatcctg ctagatcttt cggttctgct 600
gtgatcacac ataatttctc taatcactgg atcttttggg tgggcccatt catcggcgga 660
gccctggccg tgctgatcta tgattttatt ctggctccaa gatctagtga tctgactgat 720
agagtgaagg tgtggacatc cggacaggtg gaagagtatg accttgatgc tgatgatatt 780
aatagtaggg tagagatgaa gcctaaa 807
<210> 4
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gctactaact ttagcctgct gaagcaggcc ggggacgtgg aagagaaccc tggccct 57
<210> 5
<211> 717
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gtgagcaagg tcgaggagct gttcaccggc gtggtgccca tcctggtgga gctggacggc 60
gacgtgaacg gccacaagtt cagcgtgagc ggcgagggcg agggcgacgc cacctacggc 120
aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctg 180
gtgaccaccc tgacctacgg cgtgcagtgc ttcagcaggt accccgacca catgaagcag 240
cacgacttct tcaagagcgc catgcccgag ggctacgtgc aggagaggac catcttcttc 300
aaggacgacg gcaactacaa gaccagggcc gaggtgaagt tcgagggcga caccctggtg 360
aacaggatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct gggccacaag 420
ctggagtaca actacaacag ccacaacgtg tacatcatgg ccgacaagca gaagaacggc 480
atcaaggtga acttcaagat caggcacaac atcgaggacg gcagcgtgca gctggccgac 540
cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600
ctgagcaccc agagcgccct gagcaaggac cccaacgaga agagggacca catggtgctg 660
ctggagttcg tgaccgccgc cggcatcacc ctgggcatgg acgagctgta caagtaa 717
<210> 6
<211> 589
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60
ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120
atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180
tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240
ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300
attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360
ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420
gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480
aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540
cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgc 589
<210> 7
<211> 477
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca 60
gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc 120
ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca 180
acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg 240
ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg 300
ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg 360
ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct 420
tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtcctt 477
Claims (5)
1. The recombinant adeno-associated virus core plasmid is characterized in that the core plasmid carries expression frames of cre-dependent expressed aquaporin AQP1 gene and fluorescent protein reporter gene.
2. The recombinant adeno-associated virus core plasmid of claim 1, wherein a CAG promoter, a DIO element, an AQP1 gene, a 2A junction sequence, an EGFP gene, a WPRE post-transcriptional regulatory element and a polyA sequence are sequentially inserted into the ITR of the rAAV vector plasmid to construct the recombinant adeno-associated virus core pAAV-CAG-DIO-AQP 1-2A-EGFP-WPRE-pA.
3. A recombinant adeno-associated virus comprising the pAAV-CAG-DIO-AQP1-2A-EGFP-
WPRE-pA is AAV 2-retrotro serotype recombinant adeno-associated virus prepared by packaging core plasmids.
4. Use of the recombinant adeno-associated virus according to claim 3 for detecting cell-type specific neural connections at the in vivo level based on magnetic resonance imaging.
5. The use of claim 4, wherein the subject comprises cre transgenic mice and rats.
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| CN114107231A (en) * | 2021-12-13 | 2022-03-01 | 重庆大学 | Recombinant adeno-associated virus for realizing cell body labeling of whole brain postsynaptic neurons and application thereof |
| CN114921494A (en) * | 2022-04-12 | 2022-08-19 | 西北工业大学 | RNAi expression vector for Cre enzyme-dependent rAAV expression and application thereof |
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| CN113005150A (en) * | 2019-12-20 | 2021-06-22 | 复旦大学附属华山医院 | Preparation method of novel animal rapid eye movement sleep behavior disorder model |
| CN113025657A (en) * | 2021-03-05 | 2021-06-25 | 中国科学院精密测量科学与技术创新研究院 | Adeno-associated virus for bimodal imaging and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111825772A (en) * | 2020-07-30 | 2020-10-27 | 中国科学院精密测量科学与技术创新研究院 | Adeno-associated virus with variant capsid protein and uses thereof |
| CN111825772B (en) * | 2020-07-30 | 2023-10-20 | 中国科学院精密测量科学与技术创新研究院 | Adeno-associated virus with variant capsid protein and application thereof |
| CN114107231A (en) * | 2021-12-13 | 2022-03-01 | 重庆大学 | Recombinant adeno-associated virus for realizing cell body labeling of whole brain postsynaptic neurons and application thereof |
| CN114107231B (en) * | 2021-12-13 | 2023-08-18 | 重庆大学 | Recombinant adeno-associated virus for realizing whole brain postsynaptic neuron cell body marking and application thereof |
| CN114921494A (en) * | 2022-04-12 | 2022-08-19 | 西北工业大学 | RNAi expression vector for Cre enzyme-dependent rAAV expression and application thereof |
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