CN110004118A - For assisting recombinant rabies poison in the efficient reverse packing method and its application of the rAAV virus across single-stage cynapse of nerve cell - Google Patents
For assisting recombinant rabies poison in the efficient reverse packing method and its application of the rAAV virus across single-stage cynapse of nerve cell Download PDFInfo
- Publication number
- CN110004118A CN110004118A CN201910259248.2A CN201910259248A CN110004118A CN 110004118 A CN110004118 A CN 110004118A CN 201910259248 A CN201910259248 A CN 201910259248A CN 110004118 A CN110004118 A CN 110004118A
- Authority
- CN
- China
- Prior art keywords
- virus
- stage
- cynapse
- nerve cell
- raav
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000002569 neuron Anatomy 0.000 title claims abstract description 71
- 239000002574 poison Substances 0.000 title claims abstract description 55
- 231100000614 poison Toxicity 0.000 title claims abstract description 55
- 206010037742 Rabies Diseases 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000012856 packing Methods 0.000 title claims abstract description 16
- 241000700605 Viruses Species 0.000 title claims description 113
- 239000013613 expression plasmid Substances 0.000 claims abstract description 58
- 210000004027 cell Anatomy 0.000 claims abstract description 45
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 25
- 238000004806 packaging method and process Methods 0.000 claims abstract description 19
- 241000702421 Dependoparvovirus Species 0.000 claims abstract description 17
- 108091006027 G proteins Proteins 0.000 claims abstract description 12
- 108091000058 GTP-Binding Proteins 0.000 claims abstract description 12
- 239000000203 mixture Substances 0.000 claims abstract description 11
- 241000711798 Rabies lyssavirus Species 0.000 claims abstract description 9
- 239000013612 plasmid Substances 0.000 claims description 25
- 102000004190 Enzymes Human genes 0.000 claims description 23
- 108090000790 Enzymes Proteins 0.000 claims description 23
- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 claims description 22
- 238000010008 shearing Methods 0.000 claims description 22
- 238000001890 transfection Methods 0.000 claims description 7
- 108010061610 Tva receptor Proteins 0.000 claims description 3
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 claims description 2
- 241000282341 Mustela putorius furo Species 0.000 claims description 2
- 241000288667 Tupaia glis Species 0.000 claims description 2
- 230000002490 cerebral effect Effects 0.000 claims description 2
- 102000057593 human F8 Human genes 0.000 claims description 2
- 229940047431 recombinate Drugs 0.000 claims description 2
- 241000252212 Danio rerio Species 0.000 claims 1
- 210000004498 neuroglial cell Anatomy 0.000 claims 1
- 210000002856 peripheral neuron Anatomy 0.000 claims 1
- 210000002706 plastid Anatomy 0.000 claims 1
- 210000004556 brain Anatomy 0.000 abstract description 58
- 238000002156 mixing Methods 0.000 abstract description 9
- 102000030782 GTP binding Human genes 0.000 abstract description 5
- 208000003322 Coinfection Diseases 0.000 abstract description 4
- 238000013326 plasmid cotransfection Methods 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 description 17
- 230000003612 virological effect Effects 0.000 description 15
- 230000006798 recombination Effects 0.000 description 13
- 238000005215 recombination Methods 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- UHZZMRAGKVHANO-UHFFFAOYSA-M chlormequat chloride Chemical compound [Cl-].C[N+](C)(C)CCCl UHZZMRAGKVHANO-UHFFFAOYSA-M 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 230000001537 neural effect Effects 0.000 description 7
- 238000011144 upstream manufacturing Methods 0.000 description 7
- 241000699660 Mus musculus Species 0.000 description 6
- 239000003999 initiator Substances 0.000 description 6
- 238000011830 transgenic mouse model Methods 0.000 description 6
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 5
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 239000005090 green fluorescent protein Substances 0.000 description 5
- 210000000976 primary motor cortex Anatomy 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 3
- 239000002131 composite material Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 210000005036 nerve Anatomy 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000000946 synaptic effect Effects 0.000 description 3
- 230000003363 transsynaptic effect Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 210000004907 gland Anatomy 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 230000003447 ipsilateral effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002858 neurotransmitter agent Substances 0.000 description 2
- 238000012858 packaging process Methods 0.000 description 2
- 230000010412 perfusion Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108010054624 red fluorescent protein Proteins 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 208000010772 Dog disease Diseases 0.000 description 1
- 241000283070 Equus zebra Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 206010067268 Post procedural infection Diseases 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 108091005948 blue fluorescent proteins Proteins 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000003710 cerebral cortex Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- NBQNWMBBSKPBAY-UHFFFAOYSA-N iodixanol Chemical compound IC=1C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C(I)C=1N(C(=O)C)CC(O)CN(C(C)=O)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NBQNWMBBSKPBAY-UHFFFAOYSA-N 0.000 description 1
- 229960004359 iodixanol Drugs 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000002186 photoactivation Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 208000037920 primary disease Diseases 0.000 description 1
- 210000001176 projection neuron Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/106—Primate
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/40—Fish
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Environmental Sciences (AREA)
- Organic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a kind of for assisting the packing method and application of recombinant adeno-associated virus of the recombinant rabies poison in nerve cell efficiently inversely across single-stage cynapse.By the way that the expression plasmid for carrying fluorescence protein gene and TVA acceptor gene altogether and the expression plasmid for carrying rabies viruses memebrane protein (G-protein) gene are pre-blended into expression plasmid mixture; then after assisting packaging plasmid cotransfection incasing cells with recombinant adeno-associated virus, the single recombinant adeno-associated virus set having good stability is obtained by purifying.The method for being individually packaged into mixed infection after recombinant adeno-associated virus compared to different expression plasmids, it can be substantially reduced exclusive problem caused by different recombinant adeno-associated virus mixing coinfections through the invention, the novel recombinant adeno-associated virus obtained can assist recombinant rabies poison more efficiently to mark brain nervous cell and input network in the single-stage of full brain, it can obtain the more complete full brain single-stage of the specific brain area nerve cell of brain and input network.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to one kind is for assisting recombinant rabies poison efficient in nerve cell
The inversely packing method and its application of the rAAV virus across single-stage cynapse.
Background technique
Neural circuitry is very huge by quantity and the diversified nerve cell of type passes through certain mode connection group
At, be brain exercise various functions structure basis.It is more preferably geographical that the accurate Analysis of neural circuitry structure connection facilitates people
How solution brain exercises different functions.Inversely across single-stage cynapse tagging system is inverse as parsing nerve cell for recombinant rabies poison
It is widely used in neural circuitry research to the tool of single-stage input network.
Inversely across single-stage cynapse tagging system commonly uses recombinant adeno-associated virus (rAAV) as auxiliary to recombinant rabies poison at present
Virus, rAAV virus signature nerve cell effect stability are helped, and is easy to carry out genome modification.RAAV helper virus system master
Three kinds of albumen are expressed, namely for characterizing the fluorescin of helper-virus infection, for mediating recombinant rabies poison special
Property enter the TVA receptor of helper-virus infection nerve cell, and the assembling of auxiliary recombinant rabies poison and further across cynapse mark
The G-protein of note.Common method is that above-mentioned three kinds of protein gene are respectively placed in two difference rAAV matter in document report at present
Grain carrier, wherein TVA acceptor gene and fluorescence protein gene are placed in same plasmid vector, and G-protein gene is placed in another matter
Grain carrier, hybrid injection is big after the above two plasmid for carrying different expression albumen is then packaged into two kinds of rAAV viruses respectively
The specific brain area of brain simultaneously combines recombination RV virus, realizes label of the specific brain area nerve cell of brain inversely across single-stage cynapse.Wherein
The nerve cell quilt of the fluorescence registration mark of the fluorescence and recombination RV infection nerve cell of rAAV helper-virus infection nerve cell
Referred to as initiator cell, since inversely across cynapse upstream neuron does not have G-protein expression to recombination RV virus, across cynapse label
RV virus is recombinated in the neuron of upstream cannot be further across cynapse, therefore the system defines that recombination RV virus can only be inversely across one
Grade cynapse.
In addition, inversely across single-stage cynapse tagging system can be divided into applied to particular cell types nerve carefully recombinant rabies poison
The two types of born of the same parents or nonspecific cell type nerve cell.Wherein the label of particular cell types nerve cell mainly passes through
The transgenic mice reality of the auxiliary rAAV virus expressed and specific nerve cell expression Cre shearing enzyme is relied in conjunction with Cre shearing enzyme
Existing, i.e., auxiliary rAAV virus only could normal expression in a kind of nerve cell of cell type specific expression Cre shearing enzyme.It is non-
Particular cell types nerve cell label refers to i.e. effable heavy without auxiliary rAAV virus in the presence of Cre shearing enzyme
Inversely across single-stage cynapse tagging system, this method are mainly used in the nonspecific cell type nerve of target brain area carefully to group rabies viruses
The research of the full brain single-stage input network of born of the same parents.
Since different virus mixing postoperative infection cell will cause different component interaction in virus mixture or repel.
Therefore in above two rAAV helper virus mixed method three kinds of albumen (TVA, XFP, RG) cannot complete high efficient expression, and then lead
Cause recombination RV virus can not the full brain of highly efficient labeling target brain area nerve cell input network, therefore existing there are two types of rAAV auxiliary diseases
Malicious hybrid injection and the single-stage input net for combining the full brain single-stage input network of the nerve cell of recombination RV virus signature all with it
Network still has a certain distance, constrains people to the reason of the complete neural circuitry organizational structure of specific brain area nerve cell
Solution.
Aiming at the problem that above-mentioned various methods are brought or difficulty, the invention discloses one kind for assisting recombinant rabies
Poison is in the efficient reverse packing method and application of the rAAV virus across single-stage cynapse of nerve cell.The rAAV virus packing method
The method for being packaged into single rAAV virus set is amounted to based on multi-core expression plasmid premix, by the way that fluorescin base will be carried altogether
The expression plasmid of the expression plasmid and carrying rabies viruses memebrane protein (G-protein) gene of cause and TVA acceptor gene is pre-blended into
Expression plasmid mixture after then assisting packaging plasmid cotransfection incasing cells with recombinant adeno-associated virus, is obtained by purifying
The single recombinant adeno-associated virus set having good stability.It is related that recombination gland is individually packaged into compared to different expression plasmids
The method of mixed infection after virus can be substantially reduced through the invention caused by different recombinant adeno-associated virus mixing coinfections
Exclusive problem, it is thin that the novel recombinant adeno-associated virus of acquisition can assist recombinant rabies poison more efficiently to mark cerebral nerve
Born of the same parents input network in the single-stage of full brain, and then help people that the input network connection of nerve cell is more fully understood, and are people
Understanding brain working principle establishes certain structure basis.
Summary of the invention
The purpose of the invention is to provide one kind for assisting recombinant rabies poison in nerve cell efficiently inversely across list
The packing method and its application of the rAAV virus of grade cynapse.By by different auxiliary rAAV viral expression plasmids pre-mix packs at
Single rAAV virus set is inputted further combined with recombinant rabies poison label nerve cell in the more complete single-stage of full brain level
Network helps the input network and input pattern of people's better and more comprehensively understanding and cognition nerve cell.
To achieve the goals above, the invention adopts the following technical scheme:
For assisting recombinant rabies poison in the efficient reverse rAAV virus of across single-stage cynapse label of nerve cell, by with
Lower method packaging preparation: by the helper virus expression plasmid for co-expressing fluorescence protein gene and TVA acceptor gene and weight is carried
Group rabies virus G protein gene helper virus expression plasmid be pre-mixed into core expression plasmid mixture, then with rAAV
Virus auxiliary packaging plasmid cotransfection incasing cells, is further purified and obtains single rAAV virus set.
Further, it is mad that fluorescence protein gene, the helper virus expression plasmid of TVA acceptor gene and carrying recombination are co-expressed
The helper virus expression plasmid of dog viral G protein gene is mixed by mole ratio 1:0.5~1:20.
Further, the rAAV helper virus expression plasmid includes that Cre shearing enzyme such as relies on the AAV-EF1 of expression
α-DIO-GT (GFP-TVA) plasmid and AAV-EF1 α-DIO-G plasmid etc., wherein EF1 α promoter can be replaced by it is other such as
CAG, CMV, Syn etc. can be used for the promoter of nerve cell label, and GFP fluorescin element can be replaced by other can be used for
The fluorescin element of neural markers, blue fluorescent protein BFP, yellow fluorescence protein YFP, red fluorescent protein RFP etc.
Other a variety of fluorescin elements.
Further, the helper virus expression plasmid, can be in addition to that can be designed to that Cre shearing enzyme relies on expression
Be designed to Flp shearing enzyme, Dre shearing enzyme or Vika shearing enzyme rely on expression or above four kinds of shearings enzyme in any two kinds or
The helper virus expression plasmid that the combination of three kinds of shearing enzymes relies on.
Further, the helper virus expression plasmid also may be designed to the type of acellular selective expression, i.e.,
RAAV helper virus can directly express for assisting recombinant rabies poison across needed for cynapse in the presence of non-shear enzyme
TVA acceptor gene, fluorescence protein gene and G-protein gene, the system are mainly used for nerve cell non-cell type selectivity
Inversely across cynapse label.
Further, neuromechanism label or functional label element can be increased in helper virus expression plasmid.For example increase
Add fluorescin element copy number, improves the abundance of unit time inner virus expression fluorescin；It is glimmering to increase nerve cell cynapse
Signal element (such as Synaptophysin-XFP) can mark nerve cell high definition synaptic structure；Increase the heredity of the light such as Chr2
Element is learned, the excitation photoactivation of different wave length or the activity of inhibition neuron are used；Increase Gcamp calcium image-forming component, detection
Calcium ion in nerve cell activity is provided horizontal；Increase various different neurotransmitter probe members, detection nerve cell release
Neurotransmitter levels；And other elements for being possibly used for nerve cell research.
Further, the packing method of the recombination rAAV virus, the serotype of virus packaging are not limited only to 2/9 type,
Should also include other serotypes that can be used for nerve cell label, for example, 2/1,2/2,2/5 ,/2/6,2/8,2/DJ etc. it is other
Often in the serotype of neural markers.
Further, the packing method of the recombination rAAV virus can be used for zebra due to the universality of rAAV infection
The brain nervous cell and Deiter's cells, periphery of other animals such as fish, rat, cavy, ferret, tree shrew, non-human primates
The sparse highlighted scale designation of nerve cell.
By the rAAV virus that the above method is packed can be used for assisting recombinant rabies poison nerve cell efficiently inversely across
Single-stage cynapse label, is illustrated by taking the brain nervous cell of mouse as an example:
1, it is used to assist recombinant rabies poison in the nerve cell rAAV that efficiently inversely across single-stage cynapse marks acquisition
Viral stereotaxical injection to the specific brain area of mouse brain, expression injects recombinant rabies in identical injection site after a certain period of time
Poison, inversely across single-stage cynapse marks the single-stage of target brain area nerve cell to recombinant rabies poison under the auxiliary of auxiliary rAAV virus
Input network；
2, the further whole br ain slices of across the cynapse label result of the recombinant rabies poison obtained in step 1 are imaged, can be obtained
It obtains the number of target brain area starting nerve cell and the full brain single-stage input nerve cell of initiator cell is distributed and number, to obtain
It obtains single-stage of the target brain area nerve cell within the scope of full brain efficiently across cynapse label and inputs network.
Compared with prior art, the present invention has the following advantages and beneficial effects:
1, packing method is relatively easy and save the cost, and compared to past method, this method only needs to pack primary disease
Poison saves human cost and economic cost in packaging process.The single rAAV virus set that packaging obtains infects mind
Fine through cell stability, the experimental result otherness that different batches experiment obtains is smaller.
2, compared to past labeling method, under same infectious condition, novel two kinds of helper virus expression plasmids are first mixed
The mode for being packaged into single rAAV virus set after conjunction altogether can help more efficiently across the cynapse label upstream of recombinant rabies poison defeated
Enter the more nerve cell of brain area number, that is, obtains more fully full brain single-stage input network.And labelling experiment is easy to operate
It is easy, it is only necessary to primary single rAAV virus set is injected, it is greatly simple without operations such as other additional virus mixing
Change experiment flow, reduces human factor bring error during cumbersome experimental implementation.
Detailed description of the invention
Fig. 1 is auxiliary viral expression plasmids AAV-EF1 α-DIO-GT and expression plasmid AAV-EF1 α-DIO-G and expression
The recombinant rabies poison of RFP (Dsred) carries related gene structural schematic diagram.
Fig. 2 is two kinds of helper virus expression plasmid AAV-EF1 α-DIO-GT and expression plasmid AAV-EF1 α-DIO-G (RG)
The process of the single rAAV virus set of pre-mix pack.
Fig. 3 is auxiliary rAAV virus and the malicious injection time flow chart of recombinant rabies.
Histogram is two kinds of auxiliary rAAV viral expression plasmids AAV-EF1 α-DIO-GT and expression plasmid AAV- on the left of Fig. 4
EF1 α-DIO-G is pre-mixed gained rAAV helper virus (cAAV-DIO-GT/RG) with 1:2 ratio and infuses in conjunction with recombinant rabies poison
Penetrate the number of Thy1-cre transgenic mice brain primary motor cortex label initiator cell.Histogram is two kinds auxiliary on the right side of Fig. 4
RAAV viral expression plasmids AAV-EF1 α-DIO-GT and expression plasmid AAV-EF1 α-DIO-G is helped to be packaged into rAAV virus respectively so
Afterwards with 1:2 ratio mixing (AAV-DIO-GT/AAV-DIO-RG) and the identical brain area of combination recombinant rabies poison label same mouse
Initial cell number.
Histogram is that auxiliary rAAV viral (cAAV-DIO-GT/RG) combines recombinant rabies poison to inject Thy1- on the left of Fig. 5
Cre transgenic mice brain primary motor cortex marks the total number of full brain input nerve cell and the ratio of initiator cell across cynapse
Value.Right side histogram is that auxiliary rAAV viral (AAV-DIO-GT/AAV-DIO-RG) combines recombinant rabies poison to inject Thy1-
Cre transgenic mice brain primary motor cortex marks the total number of full brain input nerve cell and the ratio of initiator cell across cynapse
Value.
Fig. 6 is that auxiliary rAAV viral (cAAV-DIO-GT/RG) combines recombinant rabies poison to inject Thy1-cre transgenosis
The multiple brain area input results of the full brain of across the cynapse label of mouse brain primary motor cortex and auxiliary rAAV virus (AAV-DIO-GT/
AAV-DIO-RG the comparison of corresponding across the cynapse label result of recombinant rabies poison) is combined.
Specific embodiment
In order to make that the present invention announces for assisting recombinant rabies poison in efficient reverse across the single-stage cynapse mark of nerve cell
Design concept, technical solution and a variety of advantages of the rAAV virus system of note are more readily understood, we are real by attached drawing and part
Case is applied, the present invention is described in more details.It should be noted that specific embodiment mentioned below is only used for solving
The present invention is released, but cannot be understood to the limitation to application of the invention.
Embodiment 1
For assisting the mouse brain particular cell types nerve cell efficiently reverse packet of the rAAV virus across single-stage cynapse
Dress method and compliance test result.Particular cell types nerve cell label is mainly by by helper virus expression plasmid in the present embodiment
It is designed to that Cre shearing enzyme relies on to express and combine the transgenic mice of particular cell types nerve cell expression Cre shearing enzyme real
It is existing.Main implementation steps are as follows:
1, plasmid combinations screening system
It is to co-express green fluorescent protein (GFP) and TVA receptor and express the helper virus plasmid combinations of G-protein
Example, i.e. AAV-EF1 α-DIO-GT plasmid and AAV-EF1 α-DIO-G plasmid (come from Wuhan Shu Mi brain science Technology Co., Ltd.).
Helper virus expression plasmid carry fluorescence protein gene can be green fluorescence protein gene or other fluorescence protein genes,
As long as in principle helper virus expression fluorescin can with recombinant rabies poison entrained by fluorescin fluorescence microscopy at
It can obviously be distinguished as in.
2, the packaging of reverse across the single-stage cynapse auxiliary rAAV virus of particular cell types nerve cell
The representative plasmid combinations filtered out in step 1 are expanded respectively, obtain the plasmid of sufficient amount.It will
Plasmid after amplification is pre-blended into core plasmid mixture using different mixing proportion as specific embodiment.
1) by AAV-EF1a-DIO-GT expression plasmid and AAV-EF1a-DIO-G expression plasmid with molar ratio 1:2 (wherein
GT is that ratio 1) is pre-mixed and obtains core expression plasmid mixture.
2) 293T transfects the culture of cell: after the adherent 293T cell of normal growth is digested with pancreatin, according to about 1.0
×107The amount of cells is seeded in the Tissue Culture Dish of 15cm.Then at 37 DEG C, the CO of concentration 5%2It is cultivated about in incubator
12-18h can be transfected when cell density reaches the 80%-90% of entire culture dish, and 2h replaces fresh no antibiosis before transfecting
The culture medium of element.
3) preparation of transfection composite: first determining rotaring redyeing system when transfection, by taking the cell concentration of 5 × 15cm plate as an example, turns
The system of dye is generally 5ml, including 85 μ g of AAV expression plasmid, helper plasmid (being purchased from agilent company) (pHelper) 85 μ g,
Transfection reagent PEI and dilution about the 4ml PBS of AAV capsid plasmid (pRC) 85 μ g, 765 μ l；When preparing transfection composite,
It first takes the PBS of about 4ml in the centrifuge tube of 15ml, three plasmid of AAV system is successively added in PBS according to quantity, mix, then add
Add the transfection reagent PEI (PEI/ total DNA=3:1) of 765 μ l, mix and is being stored at room temperature 15 minutes.
4) 293T cell transfecting: transfection composite is uniformly added in cell by 1ml/15cm plate, after mixing gently,
Transfection is completed, cell is placed on 37 DEG C, the CO of concentration 5%2It is cultivated in incubator, cell and culture is collected after about 72h
Base.
5) AAV viral purification: the cell that 72h after above-mentioned transfection is collected add a certain amount of cell pyrolysis liquid (NaCl:
150mM, Tris-Cl:100Mm, pH=8.0) liquid nitrogen or 37 DEG C multigelation 3 times, supernatant is taken after centrifugation.Further for receipts
The culture medium of collection is set and is incubated for 3h or overnight on ice with PEG-NaCl (final concentration is respectively 0.6M NaCl and 8% PEG (W/V)),
Supernatant is abandoned after centrifugation, is resuspended and is precipitated with PBS, and the precipitating of resuspension and cell lysate supernatant liquid are subjected to Iodixanol density level bands
Ultracentrifugation is spent, 40% layer of suspension is taken after centrifugation, is dialysed, is concentrated, obtains the single rAAV virus set of final finished
(cAAV-DIO-GT/RG), -80 DEG C of preservations are put.
Wherein, AAV-EF1a-DIO-GT expression plasmid and AAV-EF1a-DIO-G expression plasmid carry related gene signal
Figure is as shown in Figure 1, efficient inversely across the single-stage cynapse label auxiliary rAAV disease of both plasmid pre-mix pack recombinant rabies poison
The packaging process of poison is as shown in Figure 2.Similarly, in nonspecific cell type nerve cell, inversely across single-stage cynapse system assists rAAV
In virus packaging preparation process, two kinds of auxiliary rAAV virus packaging plasmids are substituted for non-Cre shearing enzyme and rely on expression,
Remaining packaging step is essentially identical.Furthermore in AAV-EF1a-DIO-GT expression plasmid entrained GFP sequence can be replaced by it is other all
Such as RFP perhaps the corresponding recombinant rabies poison of BFP fluorescent protein sequence carry fluorescin must be replaced by GFP or
RFP, specific terms of packing and plasmid mixed proportion and above-mentioned AAV-EF1a-DIO-GT expression plasmid and AAV-EF1a-DIO-G
The hybrid packed condition of expression plasmid is almost the same.
3, for assisting the result verification of reverse across the single-stage cynapse rAAV virus of particular cell types nerve cell
The single rAAV virus set (cAAV-DIO-GT/RG) of above-mentioned acquisition or two kinds of expression plasmids are packed respectively
The rAAV virus AAV-DIO-GT/AAV-DIO-RG combination particular cell types nerve cell expression Cre shearing enzyme mixed afterwards
Transgenic mice carries out proving and comparisom.Cerebral Cortex projection neuron expression Cre shearing enzyme is selected in the present invention turns base
Because mouse (Thy1-Cre, Jackson number: 006143) is used as specific application example.The specific steps are will wrap in above-mentioned steps 2
After the helper virus expression plasmid premixing of dress preparation the single rAAV virus set cAAV-DIO-GT/RG that is packaged into or
AAV-DIO-GT/AAV-DIO-RG stereotaxical injection 80nl to Thy1-Cre mouse brain primary motor cortex M1.It is auxiliary to this
It helps rAAV expressing viral after a certain period of time, expresses red fluorescent protein RFP in the identical injection site injection 150nl of brain
(Dsred) recombinant rabies poison RV-EnvA-Dsred carries out cardiac perfusion, the experiment to mouse after RV expressing viral 9 days
Timing node process (is dissolved in phosphate buffer PBS as shown in figure 3, mouse brain sample is taken to be placed in 4% paraformaldehyde after perfusion
In) fix overnight afterwards, it then changes into 30% sucrose solution (being dissolved in PBS buffer solution), it is big to mouse after about 48-72h
Brain sample is sink to bottom in sucrose solution, carries out frozen tissue section, slice thickness 50um, to full brain sample to mouse brain sample
This is imaged and is statisticallyd analyze the neuronal quantity of initiator cell and upstream rabies viruses label and distribution, to obtain note
Number of labels (the yellow neuron that red and green marks altogether) and its single-stage of the full brain in upstream for penetrating site Original neurons are defeated
Enter network (as shown in Figure 4,5, 6).
The rAAV virus cAAV-DIO-GT/RG that helper virus expression plasmid pre-mix pack obtains as can be seen from Figure 4
The rAAV virus AAV- that the number (left side histogram) of label Original neurons mixes after packing respectively with two kinds of expression plasmids
The number (right side histogram) of the Original neurons of DIO-GT/AAV-DIO-RG label is not significantly different, and shows that this is novel
RAAV helper virus will not dramatically increase initial cell number under identical infectious condition.
Data shown in Fig. 5 are to recombinate the number and initial cell number of the neuron that rabies viruses marks full brain across cynapse
Ratio, the data can reflect the transsynaptic efficiency of auxiliary rAAV virus system auxiliary recombinant rabies poison.It can from figure
The rAAV virus cAAV-DIO-GT/RG auxiliary recombinant rabies poison that helper virus expression plasmid pre-mix pack obtains out is across prominent
The efficiency (left side histogram) of touching is significantly higher than the rAAV virus AAV-DIO-GT/ mixed after two kinds of expression plasmids are packed respectively
AAV-DIO-RG assists the transsynaptic efficiency (right side histogram) of recombinant rabies poison.Therefore, helper virus expression plasmid premixes
Auxiliary recombinant rabies poison can be significantly improved across synaptic efficiency by closing the rAAV virus that packaging obtains, and be illustrated of the present invention
The superiority of helper virus expression plasmid pre-mix pack auxiliary rAAV viral methods.
Data shown in Fig. 6 are the number for recombinating rabies viruses and marking each input brain area labeled neurons in full brain upstream across cynapse
The qualitative figure of amount.The data more intuitively reflect the transsynaptic efficiency of auxiliary rAAV virus system auxiliary recombinant rabies poison.
As can be seen from the figure the rAAV virus cAAV-DIO-GT/RG that helper virus expression plasmid pre-mix pack obtains assists recombination
Across the cynapse multiple brain areas in label upstream (the ipsilateral brain area: PFC, AI, SSP, Rt, BLA, VAL/VP, SC, MRN, SNr of rabies viruses
And opposite side brain area: PFC, Mop, AI, Rt, VAL/VP, SC, SNr, IP, SPVI) the number of neuron be above two kinds of tables
RAAV virus AAV-DIO-GT/AAV-DIO-RG auxiliary across the cynapse label of recombinant rabies poison mixed after being packed respectively up to plasmid
Result.Wherein such as ipsilateral brain area BLA, SC, MRN, SNr and opposite side brain area Mop, AI, Rt, VAL/VP, SC, SNr, IP,
Across the cynapse number of labels of SPVI brain area increases more obvious.Therefore, which qualitatively shows that helper virus expression plasmid premixes
Auxiliary recombinant rabies poison can be significantly improved across synaptic efficiency by closing the rAAV virus that packaging obtains, and further illustrate the present invention
The superiority of the helper virus expression plasmid pre-mix pack auxiliary rAAV viral methods.
In short, of the present invention for assisting the efficient inversely recombination gland related diseases across single-stage cynapse of recombinant rabies poison
Poison, can be by mixing two kinds of helper virus plasmid AAV-EF1 α-DIO-GT and AAV-EF1 α-DIO-G with certain proportion in advance
It is packaged into single rAAV virus after conjunction to gather to obtain, compared to currently used first by two kinds of different auxiliary viral expression plasmids point
The mode for not being bundled into different virus and then being used in mixed way, two kinds of helper virus plasmids first mix is packaged into single virus system afterwards
Mode can obtain better expression effect, be embodied in two kinds of helper virus plasmids first mix pack afterwards acquisition recombination it is mad
Dog disease poison across cynapse auxiliary r AAV virus it is more efficient.Since helper virus need to only inject single rAAV virus set, also without
Need different virus mixing or dilution infection and etc., greatly reduce virus packaging manpower consumption and time loss,
Increase the efficiency of rAAV virus packaging and the stability of experimental result.
The foregoing is merely illustrative of the preferred embodiments of the present invention, be to further explanation of the invention, but not to
The limitation present invention, all any equivalent replacements made within the principle and spirit of the invention, modification and improvement etc. should all include
Within protection scope of the present invention.
Claims (8)
1. for assisting recombinant rabies poison in the packaging side of the nerve cell rAAV virus that efficiently inversely across single-stage cynapse marks
Method, which is characterized in that recombinate the helper virus expression plasmid for co-expressing fluorescence protein gene and TVA acceptor gene and expression
The helper virus expression plasmid of rabies virus G protein gene is pre-mixed into core expression plasmid mixture, then sick with rAAV
The poison auxiliary common Transfection of packaging cells of packaging plasmid, is further purified and obtains single rAAV virus set.
2. according to claim 1, for assisting recombinant rabies poison in nerve cell, efficiently inversely across single-stage cynapse is marked
RAAV virus packing method, which is characterized in that the helper virus expression plasmid include Cre shearing enzyme, Flp shearing
Enzyme, Dre shearing enzyme, Vika shearing enzyme rely on the combination of any two or three of shearing enzyme in expression and these four shearing enzymes
The coexpression fluorescin of dependence and the plasmid of TVA receptor and the plasmid for expressing recombinant rabies poison G-protein gene.
3. according to claim 1, for assisting recombinant rabies poison in nerve cell, efficiently inversely across single-stage cynapse is marked
RAAV virus packing method, which is characterized in that coexpression fluorescence protein gene and TVA acceptor gene helper virus expression
The helper virus expression plasmid of plasmid and expression recombinant rabies poison G-protein gene is mixed by mole ratio 1:0.5~1:20.
4. according to claim 1, for assisting recombinant rabies poison in nerve cell, efficiently inversely across single-stage cynapse is marked
RAAV virus packing method, which is characterized in that can increase in helper virus expression plasmid other neuromechanisms label or
Functional label element.
5. according to claim 1, for assisting recombinant rabies poison in nerve cell, efficiently inversely across single-stage cynapse is marked
RAAV virus packing method, which is characterized in that virus packaging serotype be not limited only to 2/9 type, should also include 2/1,2/2,
2/5,2/6,2/8,2/DJ serotype.
6. the rAAV virus of any one of claim 1-5 the method packaging is in auxiliary recombinant rabies poison in nerve cell height
Imitate the application in inversely across single-stage cynapse label.
7. application according to claim 6, which is characterized in that the nerve cell includes cerebral neuron and neuroglia
Cell plastid, peripheral neurons and Deiter's cells.
8. application according to claim 6, which is characterized in that recombinant adeno-associated virus application includes mouse, zebra
Fish, rat, cavy, ferret, tree shrew and non-human primate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910259248.2A CN110004118B (en) | 2019-03-29 | 2019-03-29 | Packaging method and application of rAAV (rapid access Virus) for assisting recombinant rabies virus in efficient reverse cross-single-stage synapse of nerve cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910259248.2A CN110004118B (en) | 2019-03-29 | 2019-03-29 | Packaging method and application of rAAV (rapid access Virus) for assisting recombinant rabies virus in efficient reverse cross-single-stage synapse of nerve cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110004118A true CN110004118A (en) | 2019-07-12 |
| CN110004118B CN110004118B (en) | 2023-03-28 |
Family
ID=67169374
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910259248.2A Active CN110004118B (en) | 2019-03-29 | 2019-03-29 | Packaging method and application of rAAV (rapid access Virus) for assisting recombinant rabies virus in efficient reverse cross-single-stage synapse of nerve cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110004118B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110331166A (en) * | 2019-07-31 | 2019-10-15 | 中国科学院武汉物理与数学研究所 | The bis- recombinase cascade Mach-Zehnder interferometers of CRE and FLP drive in the wrong direction across monosynaptic assistant carrier and its application |
| CN112143712A (en) * | 2020-09-30 | 2020-12-29 | 中国科学院深圳先进技术研究院 | Recombinant adeno-associated virus, preparation method thereof and application thereof in antibody detection |
| CN114085872A (en) * | 2021-11-10 | 2022-02-25 | 中国科学院深圳先进技术研究院 | Construction method and application of mouse model for expressing TVA |
| 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 |
| CN114196705A (en) * | 2021-12-07 | 2022-03-18 | 中国科学院深圳先进技术研究院 | Recombinant adeno-associated virus packaging plasmid, recombinant adeno-associated virus and application thereof |
| WO2023077621A1 (en) * | 2021-11-08 | 2023-05-11 | 中国科学院深圳先进技术研究院 | Adeno-associated virus expression vector and construction, virus preparation and cell marking methods therefor |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2894718A1 (en) * | 2012-11-21 | 2014-05-30 | Circuit Therapeutics, Inc. | System and method for optogenetic therapy |
| CA3012791A1 (en) * | 2016-01-27 | 2017-08-03 | Universitat Zurich | Use of gabaa receptor modulators for treatment of itch |
| CN108359640A (en) * | 2018-01-31 | 2018-08-03 | 武汉枢密脑科学技术有限公司 | A kind of rabies viruses of the full brain area neural network structure of label, it is prepared and application |
| CN109266682A (en) * | 2018-09-29 | 2019-01-25 | 中国科学院武汉物理与数学研究所 | A kind of nerve cell quickly drives in the wrong direction the method and application marked across cynapse |
-
2019
- 2019-03-29 CN CN201910259248.2A patent/CN110004118B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2894718A1 (en) * | 2012-11-21 | 2014-05-30 | Circuit Therapeutics, Inc. | System and method for optogenetic therapy |
| CA3012791A1 (en) * | 2016-01-27 | 2017-08-03 | Universitat Zurich | Use of gabaa receptor modulators for treatment of itch |
| CN108359640A (en) * | 2018-01-31 | 2018-08-03 | 武汉枢密脑科学技术有限公司 | A kind of rabies viruses of the full brain area neural network structure of label, it is prepared and application |
| CN109266682A (en) * | 2018-09-29 | 2019-01-25 | 中国科学院武汉物理与数学研究所 | A kind of nerve cell quickly drives in the wrong direction the method and application marked across cynapse |
Non-Patent Citations (1)
| Title |
|---|
| 靳森: "工具病毒系统的建立及其在神经环路解析中的应用", 《中国优秀博士学位论文全文数据库》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110331166A (en) * | 2019-07-31 | 2019-10-15 | 中国科学院武汉物理与数学研究所 | The bis- recombinase cascade Mach-Zehnder interferometers of CRE and FLP drive in the wrong direction across monosynaptic assistant carrier and its application |
| CN110331166B (en) * | 2019-07-31 | 2023-02-03 | 中国科学院武汉物理与数学研究所 | CRE and FLP double-recombinase cascade controlled retrograde single-synapse crossing auxiliary vector and application thereof |
| CN112143712A (en) * | 2020-09-30 | 2020-12-29 | 中国科学院深圳先进技术研究院 | Recombinant adeno-associated virus, preparation method thereof and application thereof in antibody detection |
| CN112143712B (en) * | 2020-09-30 | 2022-11-01 | 中国科学院深圳先进技术研究院 | Recombinant adeno-associated virus, preparation method thereof and application thereof in antibody detection |
| WO2023077621A1 (en) * | 2021-11-08 | 2023-05-11 | 中国科学院深圳先进技术研究院 | Adeno-associated virus expression vector and construction, virus preparation and cell marking methods therefor |
| CN114085872A (en) * | 2021-11-10 | 2022-02-25 | 中国科学院深圳先进技术研究院 | Construction method and application of mouse model for expressing TVA |
| CN114196705A (en) * | 2021-12-07 | 2022-03-18 | 中国科学院深圳先进技术研究院 | Recombinant adeno-associated virus packaging plasmid, recombinant adeno-associated virus 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110004118B (en) | 2023-03-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110004118A (en) | For assisting recombinant rabies poison in the efficient reverse packing method and its application of the rAAV virus across single-stage cynapse of nerve cell | |
| Schwarz et al. | Viral-genetic tracing of the input–output organization of a central noradrenaline circuit | |
| Saunders et al. | Novel recombinant adeno-associated viruses for Cre activated and inactivated transgene expression in neurons | |
| Callaway et al. | Monosynaptic circuit tracing with glycoprotein-deleted rabies viruses | |
| Rowland et al. | Transgenically targeted rabies virus demonstrates a major monosynaptic projection from hippocampal area CA2 to medial entorhinal layer II neurons | |
| Tang et al. | Faithful expression of multiple proteins via 2A-peptide self-processing: a versatile and reliable method for manipulating brain circuits | |
| Weible et al. | Transgenic targeting of recombinant rabies virus reveals monosynaptic connectivity of specific neurons | |
| CN109880807B (en) | Packaging method of nerve cell sparse high-brightness labeled recombinant adeno-associated virus and application thereof | |
| CN107988261A (en) | The method for producing IPS cells | |
| WO2020097121A1 (en) | Artificial expression constructs for selectively modulating gene expression in excitatory cortical neurons | |
| Suzuki et al. | Multiplex neural circuit tracing with G-deleted rabies viral vectors | |
| Aboody-Guterman et al. | Green fluorescent protein as a reporter for retrovirus and helper virus-free HSV-1 amplicon vector-mediated gene transfer into neural cells in culture and in vivo | |
| Düring et al. | Fast retrograde access to projection neuron circuits underlying vocal learning in songbirds | |
| Fenno et al. | A guide to creating and testing new INTRSECT constructs | |
| Jackson et al. | Genetic manipulation of myoblasts and a novel primary myosatellite cell culture system: comparing and optimizing approaches | |
| Pouchelon et al. | A versatile viral toolkit for functional discovery in the nervous system | |
| Kalina et al. | Retrovirus-mediated in vitro gene transfer into chicken male germ line cells | |
| Luebke et al. | Adenoviral and AAV-mediated gene transfer to the inner ear: role of serotype, promoter, and viral load on in vivo and in vitro infection efficiencies | |
| CN108359640A (en) | A kind of rabies viruses of the full brain area neural network structure of label, it is prepared and application | |
| Veres et al. | CaMKIIα promoter-controlled circuit manipulations target both pyramidal cells and inhibitory interneurons in cortical networks | |
| Zhang et al. | Fiber modifications enable fowl adenovirus 4 vectors to transduce human cells | |
| Shi et al. | A new anterograde trans-synaptic tracer based on Sindbis virus | |
| Cardona et al. | Astrocyte infection is required for retrovirus-induced spongiform neurodegeneration despite suppressed viral protein expression | |
| Jin et al. | Long-term labeling and imaging of synaptically-connected neuronal networks in vivo using nontoxic, double-deletion-mutant rabies viruses | |
| CN116200429A (en) | Recombinant adeno-associated virus composition and application thereof in precise sparse marking |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |