CN114107231B - Recombinant adeno-associated virus for realizing whole brain postsynaptic neuron cell body marking and application thereof - Google Patents

Recombinant adeno-associated virus for realizing whole brain postsynaptic neuron cell body marking and application thereof Download PDF

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CN114107231B
CN114107231B CN202111520739.1A CN202111520739A CN114107231B CN 114107231 B CN114107231 B CN 114107231B CN 202111520739 A CN202111520739 A CN 202111520739A CN 114107231 B CN114107231 B CN 114107231B
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fluorescent protein
recombinant adeno
associated virus
cell
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CN114107231A (en
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孙佩
汪萌
刘珂
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Chongqing University
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Abstract

The invention discloses a method for efficiently realizing the marking of the cell bodies of a plurality of cell types postsynaptic neurons in the whole brain range by constructing a fluorescent protein element which is mediated by a specific type promoter and is coupled with a nuclear localization sequence H2B into a double-chain vector and then packaging the double-chain vector into high-titer serotype 1 recombinant adeno-associated virus. Compared with the use of dyes and other recombinant adeno-associated viruses to label only axon-projected nerve fibers, the invention can directly label neuron cell bodies and has higher labeling brightness and efficiency. Compared with neurotropic viruses, the marker has lower toxicity and higher safety, and can mediate the long-time stable expression of the target gene. The invention is applicable not only to mechanical manual sectioning, but also to automatic or semi-automatic whole brain imaging systems for displaying markers of postsynaptic neuron cell bodies. Furthermore, by combining a whole brain imaging system, the whole distribution of cell bodies of cell postsynaptic neurons in a specific brain region and a brain region at the whole brain level and the distribution of a plurality of brain regions and sub-brain regions in the whole brain range can be displayed more intuitively at the three-dimensional level, and the difference of the regulation and control intensity of different experimental individuals, different brain regions and different cell types in the same brain region on different brain regions or different types of postsynaptic neurons in an output loop can be displayed and compared in the same sample.

Description

Recombinant adeno-associated virus for realizing whole brain postsynaptic neuron cell body marking and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a recombinant adeno-associated virus, application thereof and a labeling preparation of postsynaptic neuron cell bodies, and particularly relates to a recombinant adeno-associated virus labeling preparation for realizing efficient labeling of various cell postsynaptic neuron cell bodies in the whole brain range.
Background
The nerve loop is the basis of the structure and function of the brain nervous system, and consists of a nerve input loop and a nerve output loop, wherein a specific type of nerve cells or nerve cell groups in the nerve loop receive information input (input loop) of multiple types of nerve cells in other brain regions upstream through a plurality of dendritic spines on dendrites, and after information integration is carried out on cell bodies, the information is further transmitted to the cortex and a plurality of brain regions under the cortex (output loop) through axons of length Cheng Toushe. Therefore, the analysis of the nerve input and output loops in the deep system is helpful for understanding the structure composition of the brain and the mechanism of information processing, and provides scientific basis for the diagnosis and treatment of brain diseases. Rather, a highly visualized study of the neural output loop will help us understand the connection between the targeted brain region and numerous projected brain regions downstream, between different cell types within each brain region, between specific types of neurons, and thus understand the mechanisms how the brain functions at different scales at the mesoscale (brain region-brain region) and at the microscopic scale (neuronal presynaptic-postsynaptic).
The tools currently commonly used to achieve specific brain region output loop markers are mainly of the two general classes of conventional neuropracers (Classical neural tracers) and Viral tracers (Viral tracers). The former is marked by physical diffusion (active or passive transportation), which is the earliest and most classical method applied to forward marking of nerve loops, and common forward nerve tracers mainly comprise biotinylation dextran amine (Biotinylated dextran amine, BDA), bean leukolectin (Phaseolus vulgaris-leucoaglutinin, PHA-L) and the like; the latter realizes marking in an infection mode, has wider application and higher efficiency, has flexible and changeable forms, can design various vectors according to requirements, and can be further divided into two types of recombinant adeno-associated virus (rAAV) and neurotropic virus (Neurotropic virus) mediated marking systems according to infection characteristics. The specific implementation strategies include the following:
1. injecting BDA or PHA-L coupled with a fluorescent dye (such as Alexa Fluor family) into a specific brain region;
2. injecting a fluorescent protein fragment (such as AAV-EGFP) containing normal expression or a fluorescent protein fragment (such as AAV-FLEX-EGFP or AAV-DIO-EGFP) both ends of which are wrapped by a recombinase recognition sequence into a specific brain region of a wild-type mouse and a transgenic mouse expressing Cre or creER (requiring tamoxifen drug induction), respectively;
3. neurotropic viruses, such as the H129 strain (H129-. DELTA.TK-tdT) of engineered herpes simplex virus type 1 (Herpes simplex virus 1), or vesicular stomatitis virus (Vesicular stomatitis virus, VSV), are injected into specific brain regions.
However, the above strategy still suffers from some drawbacks, such as using strategies 1 and 2, the axonal fibers of the marked neurons can only be observed in the brain region projected downstream, and the postsynaptic neuron cell bodies cannot be marked. Whereas the intensity of the axon fibers is much lower than the neuronal cell bodies, and the neuronal cell bodies will be easier to quantify than the axon fibers. Furthermore, the use of dye-labeled neuronal populations in strategy 1 was not cell type specific and the labeling efficiency was lower compared to recombinant adeno-associated virus. Although strategy 3 can be used to label the cell structure of postsynaptic neurons of specific cell types projected to downstream brain regions, compared with dyes and recombinant adeno-associated viruses, the neurotropic viruses used are more toxic and therefore have higher requirements on experimental operating environment. Furthermore, neurotropic viruses are not as capable of mediating long-term expression of genes as recombinant adeno-associated viruses. Therefore, the current output loop research field still lacks a tool capable of realizing the postsynaptic neuron cell body marking of various cell types in the whole brain range with high efficiency and long time expression.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide a recombinant adeno-associated virus, which can be used in a mediated labeling system to label the postsynaptic neuron cell bodies of various cell types in the whole brain. The plurality of cell types includes non-specific cell types and specific cell/subcellular types. Labelling was achieved by engineering promoter-mediated fluorescent protein elements coupled to nuclear localization sequence H2B into the form of double-stranded vectors, which were then packaged into high titers of serotype 1 double-stranded recombinant adeno-associated virus. Fluorescent protein elements coupled to nuclear localization sequence H2B can be used alone to achieve labeling of non-specific cell types of each brain region in wild-type animals without genetic background (e.g., C57BL/6J mice) (as shown in (1) of FIG. 6 of the specification); expression may also be mediated by a recombinase recognition sequence (as shown in FIG. 6 (2) of the specification) suitable for transgenic animal lines expressing various types of recombinases (e.g., cre, drug-induced CreER, dre, vCre, sCre, flp, etc.) or a tetracycline response sequence (as shown in FIG. 6 (3) of the specification), suitable for transgenic animal lines expressing the tetracycline transcriptional activator (Tetracycline transactivator, tTA), respectively. Expression may also be mediated by a combination of recombinase recognition sequences and tetracycline response sequences (as shown in FIG. 6 (4) of the specification).
In order to achieve the above purpose, the present invention provides the following technical solutions:
1. a preparation method of the recombinant adeno-associated virus comprises the following steps: the fluorescent protein element of the coupling nuclear localization sequence H2B mediated by the promoter is constructed into a double-chain vector, and then the double-chain vector is packaged into the recombinant adeno-associated virus by packaging cells.
The recombinant adeno-associated virus is a high titer serotype 1 double-stranded recombinant adeno-associated virus.
Further, in the recombinant adeno-associated virus, the promoter is selected from one of a broad-spectrum expression (e.g., hSyn, EF1a, CMV, CAG, etc.) and a cell type-specific expression (e.g., caMKII mediating excitatory neuronal expression and Dlx mediating gabaergic interneuron expression and TH mediating dopaminergic neuronal expression, etc.).
Further, in the recombinant adeno-associated virus, the fluorescent protein is selected from blue fluorescent protein and derivatives thereof (e.g., BFP, EBFP, EBFP2, azurite, mTagBFP, etc.), cyan fluorescent protein and derivatives thereof (e.g., CFP, ECFP, (m) Cerulean, mTurquoise, etc.), green fluorescent protein and derivatives thereof (e.g., GFP, EGFP, mClover, emerald), yellow fluorescent protein and derivatives thereof (YFP, EYFP, (m) Citrine, (m) Venus, etc.), orange fluorescent protein and derivatives thereof (mHoneydew, mKO, mOrange, mOrange2, phean 1 (2/3/4), mcrana, etc.), red fluorescent protein and derivatives thereof (mCherry, (t) dTomato, dsRed, dsRed2, kaede, mRFP1, PHTomato, mRuby, tagRFP, mBanana, mApple, mTangerine, mStrawberry, etc.), far-red fluorescent protein and derivatives thereof (e.g., mKate 2, mMaroon1, etc.
Furthermore, in the recombinant adeno-associated virus, the fluorescent protein element coupled with the nuclear localization sequence H2B is expressed singly or is mediated by a recombinase recognition sequence and a tetracycline response sequence.
Further, in the recombinant adeno-associated virus, the recombinase recognition sequences are selected from among lox sequences recognized by Cre, variant sequences (e.g., lox N, lox 2722), and combinations of lox and variant sequences, such as loxP and loxP (LSL), loxP and lox2722 (FLEX or DIO); flp-recognized FRT sequences, variant sequences (e.g., F3, F5), and combinations of FRT and variant sequences, such as FRT and F5 (fDIO). Dre recognizes between rox sequences, between variant sequences (e.g., rox1, rox2, rox 3), and combinations of rox and variant sequences, e.g., rox1 and rox2 (dDIO). Also included are combinations between any two or more recombinase recognition sequences, such as loxP-lox2722 and FRT-F5, loxP-lox2722 and rox1-rox2, FRT-F5 and rox1-rox2, and the like.
Further, in the recombinant adeno-associated virus, the packaging cell is a HEK293T cell.
Further, the packaging is a conventional three-plasmid packaging system, namely a helper plasmid (a plasmid containing the Rep and Cap genes), an adenovirus helper plasmid, and a double-stranded plasmid mixed co-transfected HEK293T cell packaging cell line.
Further, the tetracycline responsive sequence mediated transcriptional activation tagging system may be any combination of elements of the above described promoters, fluorescent proteins and recombinase recognition sequences.
2. Use of a recombinant adeno-associated virus as defined in any one of the preceding claims for neuronal cell body markers.
Further, the recombinant adeno-associated virus is useful in labeling neuronal cell bodies that are postsynaptic neuronal cell bodies of a variety of cell types throughout the brain.
Further, the recombinant adeno-associated virus is used in neuronal cell body markers in wild type, transgenic animal strains expressing recombinant enzymes or tetracycline transcriptional activators and/or non-human primates such as rodents, mammals and the like.
Further, the rodent is a mouse, rat, guinea pig, or the like.
Further, the mammals are ferrets, tree shrews, cats, dogs, pigs, and the like.
3. A marker formulation for postsynaptic neuron cell bodies comprising the recombinant adeno-associated virus of any one of the preceding claims.
The labeling agent efficiently realizes the labeling of the postsynaptic neuron cell bodies of various cell types in the whole brain by the mediation of the recombinant adeno-associated virus.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
1. the recombinant adeno-associated virus can be used for marking the cell bodies of the postsynaptic neurons of various cell types in the whole brain, the targets which cannot be achieved by using dyes and other recombinant adeno-associated viruses except the recombinant adeno-associated virus can be achieved, and the direct marking of the cell bodies has the following advantages: (1) The brightness of the cell body of the neuron is far higher than that of the neuron fiber (the brightness of the neuron is more than 10 times), the signal is easy to capture in the observation or imaging process, the requirement on imaging parameters (such as the power of a mercury lamp or a laser) is greatly reduced, and the damage to a sample is reduced. In addition, the signal of the sample can be stored for a long time, which is more helpful for subsequent research. (2) Compared with nerve fibers, the cell bodies of the nerve cells are easier to realize quantitative research, the reliability and accuracy of the obtained experimental conclusion are greatly improved, and the intensity of the targeted brain region on the downstream projection brain region regulation can be more intuitively compared through qualitative and quantitative analysis of cell body distribution, so that the information processing and functional mechanism of a brain output loop can be more helpful to be understood. (3) The cell type of the neuron cell body of the projection brain region can be further identified by means of immunostaining and the like, and compared with nerve fibers, morphological characteristics such as the shape, the diameter and the like of the neuron cell body provide visual evidence for the selection of antibodies for staining, so that blind performance of experiments is avoided, and the experimental period is greatly shortened. (4) The method has the characteristic of high efficiency, is beneficial to researching the connection between the brain region with low efficiency marked by the prior method or strategy and the target brain region structure and function, or discovers a new projection brain region and a new projection type which cannot be marked by the prior method or strategy, and further improves the knowledge of researchers on the complexity of a brain output loop. As shown in FIG. 5 b, the present method was used to mark the corpus callosum to a certain number of postsynaptic neuron cell bodies, whereas previous methods and studies reported that only nerve fibers projected to the contralateral brain region were present in the brain region, as shown in FIG. 5c using AAV1-EF1a-EGFP markers.
2. The recombinant adeno-associated virus has lower marking immunogenicity, avoids the defect of higher toxicity of the neurotropic virus, has no high requirement on the injection environment, and improves the success rate of experiments. In addition, the recombinant adeno-associated virus of the invention can mediate the stable expression of the target gene for a long time.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:
FIG. 1 is a schematic representation of the construction of a vector for non-specific cell type markers (pscAAV-hSyn-H2B-mClover 3) according to the present invention.
FIG. 2 is the effect of the packaged recombinant adeno-associated virus on HEK293T cells, as observed with low and high magnification in bright field and fluorescence, respectively.
FIG. 3 illustrates an example of a slice imaging application of a recombinant adeno-associated virus-mediated nonspecific cell type labeling system for resolving postsynaptic neuron cell bodies in the full brain of the mouse auditory cortex.
FIG. 4 is an example of a whole brain imaging application of a recombinant adeno-associated virus-mediated nonspecific cell type labeling system for resolving postsynaptic neuron cell bodies in the whole brain of the mouse auditory cortex.
FIG. 5 is a graph selected from the representative brain slice of FIG. 4 to highlight the effect of labeling in numerous subbrain regions.
FIG. 6 is a schematic diagram of a recombinant adeno-associated viral vector for achieving efficient labeling of multiple types of cell postsynaptic neurons throughout the brain.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental methods for which specific conditions are not specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer.
Example 1
FIG. 6 is a schematic diagram of a recombinant adeno-associated viral vector for effecting efficient labeling of multiple cell types postsynaptic neurons throughout the brain, comprising a labeling system for non-specific cell types (1), mediated by recombinase recognition sequences (2) or tetracycline response sequences alone (3), and a system for effecting labeling of specific cell/subcellular types mediated by a combination of two sequences (4). Wherein the vector body portion of system (1) is a promoter-mediated fluorescent protein element coupled to nuclear localization sequence H2B; the carrier main body part of the system (2) is a fluorescent protein element which is mediated by a promoter and has coupled nuclear localization sequence H2B with two ends wrapped by a recombinase recognition sequence; the carrier body portion of system (3) is a promoter-mediated fluorescent protein element with a coupling nuclear localization sequence H2B preceded by a tetracycline response sequence; the vector body portion of system (4) is a promoter-mediated fluorescent protein element comprising a coupled nuclear localization sequence H2B of both the tetracycline response sequence and the recombinase recognition sequence.
A recombinant adeno-associated virus mediated marking system for efficiently realizing postsynaptic neuron cell bodies of non-specific cell types comprises the following steps of double-stranded vector construction, preparation of recombinant adeno-associated virus and the like:
1. construction of double-stranded vector comprising expressed genes: to demonstrate the effect of this marker system, a commercial double-stranded vector pscAAV-GFP (Addgene #32396; http:// www.addgene.org/32396 /) was selected for the construction of a vector of interest comprising an expression element. For the observation and imaging of the tagged postsynaptic neuron cell bodies, the promoter was chosen from hSyn (Human synopsin 1) which is widely expressed in neurons, and the fluorescent protein was chosen from the variant mcover3 of green fluorescent protein. The construction steps of the vector are shown in FIG. 1: firstly, synthesizing a hSyn-H2B-mClover3 fragment (a nucleotide sequence is shown as SEQ ID NO: 1) according to requirements; secondly, selecting two enzyme cutting sites of SnaBI and StuI from a template vector pscAAV-GFP, respectively treating the template vector pscAAV-GFP and hSyn-H2B-mClover3 by using two recombinases, and cutting fragments between a promoter CMV and fluorescent protein GFP from the template vector pscAAV-GFP after enzyme cutting so as to facilitate the insertion of the synthesized fragment hSyn-H2B-mClover 3; the template vector and the synthesized fragment after two enzyme treatments are connected by using ligase to obtain a double-stranded vector pscAAV-hSyn-H2B-mClover3 (the nucleotide sequence is shown as SEQ ID NO: 2) containing the expressed genes. It should be noted that the hSyn promoter may be replaced with other species such as EF1a, CMV and CAG, and the fluorescent protein mcover3 may be replaced with EGFP or other color species such as mCherry, etc. In addition, both ends of the H2B-mClover3 fragment can be mediated by recognition sequences of recombinases, such as a combination between two pairs of loxP and lox2722, FRT and F5, and rox1 and rox2, and a combination between any two or more recognition sequences of recombinases, such as loxP-lox2722 and FRT-F5, loxP-lox2722 and rox1-rox2, FRT-F5 and rox1-rox2, etc.; a tetracycline response sequence (TRE) may also be linked. The synthesis of the sequence after any combination of the promoter and the recombinase response element or the tetracycline response sequence, the construction of the vector and the preparation of the virus adopt similar steps.
2. Preparation of recombinant adeno-associated virus: the preparation of the virus was carried out according to a conventional three-plasmid packaging system, namely, helper plasmid (pAAV-RC) comprising Rep and Cap genes, adenovirus helper plasmid (pAAV-helper) and double-stranded plasmid pscAAV-hSyn-H2B-mClover3 constructed in the above 1 were mixed and co-transfected into HEK293T cells, and the specific packaging preparation process was as follows:
(1) Plasmid extraction: the plasmids used for the packaging were all extracted using Qiagen non-toxic extraction kit, required concentrations greater than 1. Mu.g/. Mu.L and the purity of the extracted plasmids was determined (A260/280. Apprxeq.1.8, A260/230> 2.0).
(2) Cell culture: HEK293T cells were digested with 0.25% trypsin to control cell density at 1X 10 7 Each 15cm Petri dish, 37℃C, 5% CO 2 The cells were cultured overnight in a cell incubator.
(3) Transfection: transfection can be performed after the cell density reached about 90%, fresh dmem+10% fbs medium was replaced 2 hours ago to prepare plasmid and PEI transfection complex with the following specifications (10×15cm dish is taken as an example): 8mL serum-free DMEM+about 0.4mL DNA (170. Mu.g double-stranded vector plasmid+170. Mu.g RC pAAV-RC+170. Mu.g pAAV-Helper plasmid) +1.6mL PEI, mixed well, and left to stand for 15 min, 1mL transfection complex per dish was added, 37℃C, 5% CO 2 The cell culture chamber is cultured overnight for 48-72 hours. After transfection, the cells are collected into a centrifuge tube together with the culture medium, and centrifuged, and culture medium supernatant and cell sediment are respectively harvested: virus in PEG8000 precipitation medium supernatant; lysing the cell pellet and detoxifying; adeno-associated virus particles obtained from cell pellet and supernatant were pooled.
(4) Virus purification, titer assay: purifying virus by iodixanol ultracentrifugation, and determining virus titer by fluorescence quantitative PCR to obtain recombinant adeno-associated virus titer greater than 1×10 13 viral genome/mL。
(5) Serotypes: in this example recombinant adeno-associated virus is packaged as 2/1 serotype.
HEK293T cells are infected by double-chain recombinant adeno-associated virus (scAAV 2/1-hSyn-H2B-mclover 3) obtained by packaging, a bright field diagram shows that the cell growth state is good, and fluorescent protein expression of strong signals can be observed under a low power mirror and a high power mirror, and the results are shown in figure 2 and indicate that the virus can be expressed normally.
Example 2
The nonspecific cell type labeling system was used to resolve postsynaptic neuron cell body distribution throughout the brain of the mouse AUD.
Living body marking: the packaged double-stranded recombinant adeno-associated virus (scAAV 2/1-hSyn-H2B-mclover 3) achieving the postsynaptic neuron cell body markers of the non-specific cell type was injected into the brain region (AP: -3.0mm, ml: -3.8mm, dv: -1.9 mm) of the Auditory cortex (AUD) of wild-type mouse in a volume of 100nL, after four weeks of viral expression. The labeled sample perfusion samples were used for manual slice imaging and whole brain continuous cut imaging, respectively.
Manual slice imaging: the post-perfused fixed labeled murine brain samples were rinsed with 0.01M PBS and dehydrated in 30% sucrose for 48-72 hours (time depending on the dehydration, judged to be the murine brain completely submerged in the bottom), the dehydrated samples were embedded using OCT frozen section embedding medium, the samples were continuously manually sectioned at 50 μm thickness using a frozen microtome (section range between Bregma +3.0mm to Bregma-7.0 mm), brain pieces were sequentially patched front to back and counterstained with 4',6-diamidino-2-phenylindole (DAPI) for 10-15 minutes, after rinsing clean, the anti-fluorescence quenching tablet was used to seal the pieces and nail polish was applied around the brain pieces for long-term storage. Brain slices containing cytoplasmic signaling were screened under a fluorescence microscope and representative brain slices were imaged.
Whole brain continuous cutting imaging: the perfused labeled mouse brain sample is embedded by Lowicryl HM20 resin, and the embedding process comprises the following steps: rinsing (rat brain was rinsed 3 times in 0.01M PBS for the first two times, 6 hours, and three times overnight), dewatering (sequentially with 50%, 75%, 95%, 100% and 100% ethanol for 2 hours per gradient), infiltration (sequentially with 50%, 75%, 100% and 100% resin for 2 hours, last three gradients for 2 hours), and polymerization (oven set at 48 ℃ C. For 8 hours). Whole brain imaging is performed by commercial bicolor fluorescence to display low light levelA tomosynthesis system (Fluorescence micro-optical sectioning tomography, fMOST) (model: biomapping 5000) was used at 0.3X0.3X2. Mu.m 3 Is achieved for a continuous imaging over about 4 days, a bicolor whole brain data containing 4,300, zhang Guanzhuang panels is acquired.
Data analysis: the obtained whole brain bi-color channel raw data were preprocessed and the green channel was projected maximum (100 μm thickness) per 50 coronal brain slices. And then respectively combining with a corresponding red channel single Zhang Guanzhuang brain slice (2 mu m thickness), and obtaining a data set of the double-color maximum projection for signal display. In order to better demonstrate the advantages of the invention, an AUD whole brain projection data set disclosed on the internet of the Allen brain institute is selectedhttps://connectivity.brain- map.org/The method comprises the steps of carrying out a first treatment on the surface of the Numbering: 146858006 After processing and then comparing with AUD bicolor whole brain data obtained by the marking system of the present invention. The specific method comprises the following steps: several bicolor coronary facial brain patches at different positions are respectively selected from the two sets of whole brain data sets and registered into an Allen standard brain atlas, and then the same brain region is selected to display the signal distribution of the two sets of data sets.
FIG. 3 is an example of a slice imaging application of a recombinant adeno-associated virus-mediated nonspecific cell type labeling system for resolving post-synaptic neuron cell bodies in the full brain of mouse AUD. 6 representative brain slices (from Bregma-0.9mm to Bregma-4.0 mm) of 50 μm thickness were selected to demonstrate the distribution of AUD in the cortex and the postsynaptic neuron cell bodies of numerous projected brain regions below the cortex. The brain regions of these cortex layers include: track cortex (ORB), anterior cingulate cortex (Anterior cingulate area, ACA), motor cortex (MO), olfactory outer cortex (ECT), temporal lobe cortex (Temporal association cortex, TEa), posterior cortex (Retrosplenial area, RSP), visual cortex (VIS), and olfactory inner layer (ENT), etc. The subcortical brain region includes: caudate nucleus (caudeocutamen, CP), basolateral amygdala (Basolateral amygdala, BLA), retrothalamic lateral nucleus (Lateral posterior nucleus of the thalamus, LP), perifascicular nucleus (Parafascicular nucleus, PF), medial knee (Medial geniculate complex, MG), and inferior colliculus (Inferior colliculus, IC), among others.
FIG. 4 is an example of a whole brain imaging application of a recombinant adeno-associated virus-mediated nonspecific cell type labeling system for resolving postsynaptic neuron cell bodies within the whole brain of a mouse AUD. Whole brain data were obtained using fmest bi-color channel imaging. Each 50 brain slices were maximum projected (100 μm thick) in the green channel and then combined with the corresponding red channel single Zhang Guanzhuang brain slice (2 μm thick) to obtain a two-color maximum projected dataset. The pictures in the dataset are sequentially arranged from front to back to show the distribution of the marked whole brain postsynaptic neuron cell bodies.
FIG. 5 is a graph selected from the representative brain slice of FIG. 4 to highlight the effect of labeling in numerous subbrain regions. To better demonstrate the advantages of the present invention, the effect of labelling numerous identical brain regions throughout the brain using the labelling system of the present invention and prior art methods (e.g. labelling using AAV-EF1 a-EGFP) are compared. Wherein FIG. a is the position of 6 representative brain slices selected, FIG. b is AUD bicolor whole brain data obtained using fMOST, obtained using the labeling system of the present invention, and FIG. c is modified whole brain data (https:// connectivity. Brain-map. Org/; number: 146858006) from the Allen brain study, published with AAV1-EF1a-EGFP labeled AUD axons. As shown in the figure, the postsynaptic cell markers achieved by the present invention have the following advantages over the axon markers: (1) higher efficiency of labeling: the cytoplasmic signal distribution of the markers of the invention is broader in numerous brain regions, such as primary motor cortex (Primary motor area, MOp), secondary motor cortex (Secondary motor area, MOs), anterior and medial Anterior regions (Anteromedial visual area, VISam), temporal lobe in combination with cortex teas, etc.; numerous brain regions under the cortex, such as caudate nucleus CP, postthalamolateral nucleus LP and postthalamocomplex (Posterior complex of the thalamus, PO) located in the dorsal thalamolateral group, lateral knee complex dorsal part (Dorsal part of the lateral geniculate complex, LGd) located in the dorsal thalamoknee group, and several subbrain regions of the visual colliculus: a motion related intermediate gray layer (Superior colliculus, motor related, intermediate gray layer; SCig), a motion related intermediate white layer (Superior colliculus, motor related, intermediate white layer; SCiw) and a motion related dark gray layer (SCdg, superior colliculus, motor related, deep gray layer; SCdg); midbrain reticulum core (Midbrain reticular nucleus; MRN), and the like. (2) The difference of the regulation and control intensity of the postsynaptic neuron signals of the AUD in each layer of the cortex can be accurately judged: the 5 th layer neurons are more in the cortex such as primary sensory cortex (Primary somatosensory area, SSp) and visual cortex VIS than in other layers, indicating that the AUD has the greatest intensity of regulation on these cortex 5 th layer neurons; in contrast, the axon distribution marked by AAV1-EF1a-EGFP is diffuse and difficult to quantitatively analyze, so that the signal intensity distribution of the axon fibers from AUD in each layer of other cortex cannot be accurately judged. (3) discovering new projected brain regions, new projection types: if the invention is used, a certain number of postsynaptic neuron cell bodies can be clearly observed in the corpus callosum, which indicates that loop connection exists between AUD and neurons of a specific type in the brain region, the degree of regulation and control of the brain region is judged according to the number of cell bodies, and the chemical components of the neurons can be identified by means of immunostaining and the like in the follow-up; while AAV1-EF1a-EGFP was used to label only nerve fibers that were projected to the contralateral cerebellum via this brain region, this was not achieved.
In general, the invention is applicable not only to mechanical manual sectioning, but also to automated or semi-automated whole brain imaging systems for displaying markers of postsynaptic neuron cell bodies. Furthermore, the invention combines with the whole brain imaging system, not only can more intuitively display the whole distribution of the specific brain region and the postsynaptic neuron cell bodies of the specific type cells in the brain region in the whole brain level and the distribution of a plurality of brain regions and sub-brain regions in the whole brain range in a three-dimensional level, but also can display and compare the differences of the regulation and control intensity of different experimental individuals, different brain regions and different cell types of the same brain region on different brain regions or postsynaptic neurons of different types in an output loop in parallel in the same sample.
Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
Sequence listing
<110> university of Chongqing
<120> recombinant adeno-associated virus for achieving whole brain postsynaptic neuron cell body labeling and use thereof
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ctacggcgtg gcctgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa 1740
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acctgcgatc tcgatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca 2280
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cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 2460
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gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 3960
agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 4020
atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 4080
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cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 4260
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aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 4500
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ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 4620
agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 4680
ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 4740
acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg 4800
cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc 4860
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Claims (8)

1. The use of a recombinant adeno-associated virus in neuronal cell body markers, wherein the neuronal cell body is a postsynaptic neuronal cell body of a plurality of cell types throughout the brain; the preparation method of the recombinant adeno-associated virus comprises the following steps: the fluorescent protein element of the coupling nuclear localization sequence H2B mediated by the promoter is constructed into a double-chain vector, and then the double-chain vector is packaged into the recombinant adeno-associated virus by packaging cells.
2. The use according to claim 1, wherein the promoter is selected from one of hSyn, EF1a, CMV, CAG, caMKII, dlx, TH.
3. The use according to claim 1, wherein the fluorescent protein is selected from the group consisting of blue fluorescent protein, cyan fluorescent protein, green fluorescent protein, yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, far-red fluorescent protein and derivatives of the above fluorescent proteins.
4. Use according to claim 1, wherein the expression of the fluorescent protein element coupled to the nuclear localization sequence H2B is expressed alone or is mediated by a recombinase recognition sequence, a tetracycline response sequence.
5. The use according to claim 1, wherein the recombinase recognition sequence is selected from the group consisting of a combination of two or more of loxP-STOP-loxP, FLEX, DIO, fDIO, dDIO, vcDIO and scDIO.
6. The use according to claim 1, wherein the packaging cell is a HEK293T cell.
7. The use according to claim 1, wherein the subject of the use is a mammal.
8. A preparation for labeling postsynaptic neuron cell bodies, comprising a recombinant adeno-associated virus, wherein the recombinant adeno-associated virus is prepared by: the fluorescent protein element of the coupling nuclear localization sequence H2B mediated by the promoter is constructed into a double-chain vector, and then the double-chain vector is packaged into the recombinant adeno-associated virus by packaging cells.
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