CN114107231A - Recombinant adeno-associated virus for realizing cell body labeling of whole brain postsynaptic neurons and application thereof - Google Patents

Recombinant adeno-associated virus for realizing cell body labeling of whole brain postsynaptic neurons and application thereof Download PDF

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CN114107231A
CN114107231A CN202111520739.1A CN202111520739A CN114107231A CN 114107231 A CN114107231 A CN 114107231A CN 202111520739 A CN202111520739 A CN 202111520739A CN 114107231 A CN114107231 A CN 114107231A
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recombinant adeno
associated virus
fluorescent protein
cell
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CN114107231B (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 marking postsynaptic neuron cell bodies of various cell types in the whole brain range by constructing a double-stranded vector by fluorescent protein elements mediated by specific types of promoters and coupled with a nuclear localization sequence H2B and then packaging the double-stranded vector into serotype 1 recombinant adeno-associated virus with high titer. Compared with the method that only nerve fibers projected by axons can be marked by using dyes and other recombinant adeno-associated viruses, the method can directly mark neuron soma and has higher marking brightness and efficiency. Compared with neurotropic virus, the marker of the invention has the advantages of lower toxicity, higher safety and capability of mediating the long-term stable expression of the target gene. The invention is not only suitable for mechanical and manual slicing, but also suitable for an automatic or semi-automatic whole brain imaging system for displaying the mark of the cell body of the postsynaptic neuron. Furthermore, the method can more intuitively display the whole distribution of the cell bodies of the postsynaptic neurons in a specific brain area and a brain area at the whole brain level and the distribution of a plurality of brain areas and sub-brain areas in the whole brain range at the three-dimensional level by combining a whole brain imaging system, and also display and compare the difference of the regulation and control strength of different experimental individuals, different brain areas and different cell types in the same brain area on different brain areas or different types of postsynaptic neurons in an output loop in parallel in the same sample.

Description

Recombinant adeno-associated virus for realizing cell body labeling of whole brain postsynaptic neurons 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 marker preparation of postsynaptic neuron cell bodies, and particularly relates to a marker preparation of the recombinant adeno-associated virus, which is used for realizing efficient marking of the postsynaptic neuron cell bodies of various types of cells in the whole brain range.
Background
The nerve loop is the basis of the structure and the function of a brain nervous system and consists of a nerve input loop and an output loop, a specific type of neuron or a neuron group in the nerve loop receives the information input (input loop) of a plurality of types of neurons in other upstream brain areas through a plurality of dendritic spines on dendrites, and after information integration is carried out on cell bodies, the information is further transmitted to a plurality of brain areas (output loops) below the cortex and the cortex through long-distance projected axons. Therefore, the deep and systematic analysis of neural input and output loops is helpful for understanding the structural composition of the brain and the mechanism of information processing, and provides scientific basis for the diagnosis and treatment of brain diseases. Rather, the exploration of the high visualization of the neural output circuits will help us to understand the connection relationships between the targeted brain regions and the downstream numerous projected brain regions, between different cell types within each brain region, and between specific types of neurons, and further understand the mechanisms how the brain functions at different scales, such as the mesoscopic level (brain region-brain region) and the microscopic level (pre-synaptic-post synaptic to neurons).
The tools currently used to achieve specific brain region export loop markers are mainly the two major categories of traditional neural tracers (classic neural tracers) and Viral tracers (Viral tracers). The former method of labeling by physical diffusion (active or passive transport) is the earliest and most classical method applied to the neural circuit forward labeling, and the commonly used forward nerve tracers mainly include Biotinylated glucosamine (BDA) and Phaseolus vulgaris-leucocyte agglutinin (PHA-L); the latter realizes marking in an infection mode, has wider application and higher efficiency, can design various vectors according to the requirements in flexible and variable forms, and can be further divided into two types of Recombinant adeno-associated virus (rAAV) and Neurotropic virus (Neurotropic virus) mediated marking systems according to the 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 fluorescent protein fragments (such as AAV-EGFP) containing normal expression or fluorescent protein fragments (such as AAV-FLEX-EGFP or AAV-DIO-EGFP) with both ends wrapped by recombinase recognition sequences into specific brain regions of wild mice and transgenic mice expressing Cre or creER (requiring tamoxifen drug induction);
3. a neurotropic virus, such as an H129 strain (H129- Δ TK-tdT) of a modified Herpes simplex virus type 1 (Herpes simplex virus 1), or Vesicular Stomatitis Virus (VSV) is injected into a specific brain region.
However, the above strategies still have some disadvantages, such as that axonal fibers of labeled neurons can only be observed in the brain region projected downstream using strategies 1 and 2, and postsynaptic neuron soma cannot be labeled. Whereas axonal fibers are much less bright than neuronal cell bodies and are more easily quantified than axonal fibers. In addition, the neuron population labeled with the dye in strategy 1 has no cell type specificity and is less efficient for labeling than recombinant adeno-associated virus. Strategy 3 can mark the soma structure of the postsynaptic neurons projecting from specific cell types to the downstream brain region, but compared with dye and recombinant adeno-associated virus, the toxicity of the used neurotropic virus is higher, and thus the requirement on experimental operating environment is higher. Furthermore, neurotropic viruses do not mediate long-term gene expression as do recombinant adeno-associated viruses. Therefore, the current field of output loop research still lacks a tool which can be expressed efficiently and for a long time and can realize cell body labeling of postsynaptic neurons of various cell types in the whole brain.
Disclosure of Invention
In view of the above, the present invention provides a recombinant adeno-associated virus, which can be applied in a mediated labeling system, and can be used to label the cell bodies of various cell types of post-synaptic neurons in the whole brain. The various cell types include non-specific cell types and specific cell/subcellular types. Labeling was achieved by engineering the promoter-mediated fluorescent protein element coupled to the nuclear localization sequence H2B into a double-stranded vector form, which was then packaged into high titer serotype 1 double-stranded recombinant adeno-associated virus. The fluorescent protein element coupled with the nuclear localization sequence H2B can be used independently, so that the labeling of non-specific cell types in each brain region can be realized in wild animals without genetic background (such as C57BL/6J mice) (as shown in (1) in the attached figure 6 of the specification); the expression can also be mediated by recombinase recognition sequences (shown as (2) in the attached figure 6 of the specification) or Tetracycline response sequences (shown as (3) in the attached figure 6 of the specification), wherein the former is suitable for transgenic animal strains expressing various types of recombinases (such as Cre, drug-induced CreER, Dre, vccre, sccre, Flp and the like), and the latter is suitable for transgenic animal strains expressing Tetracycline transcriptional activator (tTA). Alternatively, expression may 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 purpose, the invention provides the following technical scheme:
1. a recombinant adeno-associated virus is prepared by the following steps: the fluorescent protein element of the nuclear localization sequence H2B is coupled through the mediation of a promoter to construct a double-stranded vector, and then the double-stranded vector is packaged into the recombinant adeno-associated virus by a packaging cell.
The recombinant adeno-associated virus is serotype 1 double-stranded recombinant adeno-associated virus with high titer.
Furthermore, in the recombinant adeno-associated virus, the promoter is selected from one of promoters with broad-spectrum expression (such as hSyn, EF1 alpha, CMV, CAG and the like) and cell type specific expression (such as CaMKII for mediating excitatory neuron expression, Dlx for mediating GABAergic interneuron expression, TH for mediating dopaminergic neuron expression and the like).
Further, in the recombinant adeno-associated virus, the fluorescent protein is selected from blue fluorescent protein and its derivatives (e.g., BFP, EBFP2, Azurite, mTagBFP, etc.), cyan fluorescent protein and its derivatives (e.g., CFP, ECFP, (m) Cerulean, mTurquuose, etc.), green fluorescent protein and its derivatives (e.g., GFP, EGFP, mCloror, Emerald), yellow fluorescent protein and its derivatives (YFP, EYFP, (m) Citrine, (m) Venus, etc.), orange fluorescent protein and its derivatives (mHoneyde, mKO, mOrange2, pHOran1(2/3/4), mBanana, etc.), red fluorescent protein and its derivatives (mCherry, (t) atomic, DsRed2, Kaede, mRFP1, matTopho, Rubfy, TagRg, Bamnaant, mT, mOsage, mRed 1, etc.), and its fluorescent protein and its derivatives (mRed 1).
Furthermore, in the recombinant adeno-associated virus, the expression of the fluorescent protein element coupled with the nuclear localization sequence H2B is independent expression or mediated expression by a recombinase recognition sequence and a tetracycline response sequence.
Further, in the recombinant adeno-associated virus, the recombinase recognition sequence is selected from the group consisting of lox sequences recognized by Cre, variant sequences (e.g., lox N, lox2722), and a combination of lox and variant sequences, such as loxP and loxP (LSL), loxP and lox2722(FLEX or DIO); flp recognizes between FRT sequences, between variant sequences (e.g., F3, F5), and combinations of FRT and variant sequences, such as FRT and F5 (fDIO). Between the rox sequences recognized by Dre, between variant sequences (e.g., rox1, rox2, rox3), and combinations of rox and variant sequences, such as rox1 and rox2 (dDIO). Also included are combinations of 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 helper plasmids (plasmids containing Rep and Cap genes), adenovirus helper plasmids and double-stranded plasmids are mixed to transfect a HEK293T cell packaging cell line.
Further, the tetracycline response sequence-mediated transcriptional activation tagging system can be combined with any of the elements of the promoter, fluorescent protein, and recombinase recognition sequences described above.
2. Use of any of the recombinant adeno-associated viruses described above for neuronal cell body labeling.
Further, the recombinant adeno-associated virus is applied to the marking of neuron cell bodies, wherein the neuron cell bodies are postsynaptic neuron cell bodies of various cell types in the whole brain range.
Further, the recombinant adeno-associated virus is applied to neuron soma marking, and the applied object is wild animals such as rodents and mammals, transgenic animal strains expressing recombinase or tetracycline transcriptional activator and/or nonhuman primates.
Further, the rodent is a mouse, a rat, a guinea pig or the like.
Further, the mammals include ferrets, tree shrews, cats, dogs, pigs, and the like.
3. A labeled preparation of a cell body of a postsynaptic neuron, comprising the recombinant adeno-associated virus according to any one of the above.
The marking preparation is mediated by the recombinant adeno-associated virus, and can efficiently realize the marking of the cell bodies of the postsynaptic neurons of various cell types in the whole brain range.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
1. the recombinant adeno-associated virus can mark the cell bodies of the postsynaptic neurons of various cell types in the whole brain range, and the target which cannot be realized by using dyes and other recombinant adeno-associated viruses except the recombinant adeno-associated virus in the past is completed, and the direct marking of the cell bodies has the following advantages: (1) the cell brightness of the neuron is far higher than that of the neuron fiber (more than 10 times of brightness), signals can be easily captured in the observation or imaging process, the requirements on imaging parameters (such as power of a mercury lamp or a laser and the like) are greatly reduced, and the damage to a sample is reduced. In addition, the signal of the sample can be preserved for a long time, and the subsequent research is facilitated. (2) Compared with nerve fibers, the cell body of the neuron can realize quantitative research more easily, the reliability and the accuracy of the obtained experimental conclusion are greatly improved, and the strength of the control of the targeted brain region on the downstream projection brain region can be compared more intuitively through qualitative and quantitative analysis of cell body distribution, so that the understanding of information processing and function mechanism of a brain output loop is facilitated. (3) The cell types of the neuron cells in the projection brain region can be further identified by means of immunostaining and the like, and compared with the nerve fibers, the morphological characteristics such as the shape, the diameter and the like of the neuron cells provide visual evidence for the selected antibodies for dyeing, thereby avoiding the blind performance of the experiment and greatly shortening the experiment period. (4) The method has the characteristic of high efficiency, is beneficial to researching the relation between the structure and the function of the brain area marked with low efficiency by using the traditional method or strategy, or discovering a new projection brain area and a new projection type which cannot be marked by the traditional method or strategy, and further improves the understanding of researchers to the complexity of the brain output loop. While the calluses were labeled to a certain number of postsynaptic neuronal cells using the present method, as shown in b of FIG. 5, previous methods and studies reported that only nerve fibers projecting to the contralateral brain region were present in this brain region, as shown in c of FIG. 5 using AAV1-EF1a-EGFP labeling.
2. The recombinant adeno-associated virus is used for marking, the immunogenicity is low, the defect of high toxicity caused by using the neurotropic virus is avoided, the injection environment is not required to be high, and the success rate of the experiment is improved. In addition, the recombinant adeno-associated virus of the present invention can mediate stable expression of a target gene for a long period of time.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a schematic diagram of the construction of the vector for non-specific cell type marker of the present invention (pscAAV-hSyn-H2B-mClover 3).
FIG. 2 shows the effect of the packaged recombinant adeno-associated virus on HEK293T cells observed in bright field and fluorescence under low power and high power respectively.
FIG. 3 is an example of a slice imaging application of a recombinant adeno-associated virus-mediated non-specific cell type labeling system for the resolution of postsynaptic neuronal cell bodies in the whole brain range of the mouse auditory cortex.
FIG. 4 is an example of a whole brain imaging application of a recombinant adeno-associated virus-mediated non-specific cell type labeling system for the resolution of postsynaptic neuronal cell bodies in the whole brain range of the mouse auditory cortex.
FIG. 5 is a selection of the representative brain slice of FIG. 4 to highlight the effect of the markers in a plurality of sub-brain regions.
FIG. 6 is a schematic diagram of a recombinant adeno-associated viral vector for achieving high-efficiency labeling of postsynaptic neuronal cell bodies of multiple cell types 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 procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturers.
Example 1
FIG. 6 is a schematic diagram of a recombinant adeno-associated virus vector for achieving high efficiency in labeling of postsynaptic neuronal cell bodies of various types of cells all over the brain, including a labeling system for non-specific cell types (1), a system that can achieve labeling of specific cell/sub-cell types mediated by recombinase recognition sequences (2) or tetracycline response sequences alone (3), and a combination of both sequences (4). Wherein the vector body part of the system (1) is a fluorescent protein element mediated by a promoter and coupled with a nuclear localization sequence H2B; the vector main body part of the system (2) is a fluorescent protein element which is mediated by a promoter, wrapped by recombinase recognition sequences at two ends and coupled with the nuclear localization sequence H2B; the vector main body part of the system (3) is a fluorescent protein element which is mediated by a promoter, is provided with a tetracycline response sequence and is coupled with a nuclear localization sequence H2B; the vector body part of the system (4) is a fluorescent protein element mediated by a promoter and simultaneously containing a coupled nuclear localization sequence H2B of a tetracycline response sequence and a recombinase recognition sequence.
A recombinant adeno-associated virus mediated labeling system for efficiently realizing non-specific cell type postsynaptic neuron cell bodies comprises the processes of double-chain vector construction, recombinant adeno-associated virus preparation and the like, and comprises the following steps:
1. construction of double-stranded vector containing expressed Gene: to demonstrate the effect of this marker system, a commercial double-stranded vector, pscAAV-GFP (Addgene # 32396; http:// www.addgene.org/32396/), was chosen for construction of the vector of interest containing the expression elements. For observation and imaging of labeled postsynaptic neuron cell bodies, the promoter was selected for hsin (Human synapsin 1) which is widely expressed in neurons, and the fluorescent protein was selected for the variant of green fluorescent protein mClover 3. The construction steps of the vector are shown in FIG. 1: firstly, synthesizing hSyn-H2B-mClover3 fragment (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 processing the template vectors pscAAV-GFP and hSyn-H2B-mClover3 by using two recombinant enzymes, and cutting a fragment between a promoter CMV and a fluorescent protein GFP from the template vector pscAAV-GFP after enzyme cutting so as to be beneficial to the insertion of a synthesized fragment hSyn-H2B-mClover 3; the template vector treated by the two enzymes and the synthetic fragment are connected by using ligase to obtain a double-stranded vector pscAAV-hSyn-H2B-mClover3 (nucleotide sequence is shown as SEQ ID NO: 2) containing an expression gene. It should be noted that hSyn promoter can be exchanged for other such as EF1 α, CMV and CAG, and fluorescent protein mClover3 can also be exchanged for EGFP or other color species, such as mCherry, etc. In addition, both ends of the H2B-mClorover 3 fragment can be mediated by recognition sequences of recombinase, such as the combination between two pairs of loxP and lox2722, between FRT and F5 and between rox1 and rox2, and the combination between any two or more recognition sequences of recombinase, such as loxP-lox2722 and FRT-F5, loxP-lox2722 and rox1-rox2, FRT-F5 and rox1-rox 2; tetracycline Responsive Sequences (TREs) may also be attached. The synthesis of the sequence, the construction of the vector and the preparation of the virus are carried out by adopting similar steps after the promoter is combined with a recombinase response element or a tetracycline response sequence at will.
2. Preparation of recombinant adeno-associated virus: the virus was prepared according to the conventional three-plasmid packaging system, i.e., helper plasmid (pAAV-RC) containing 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 packaging were extracted using the Qiagen toxin-free extraction kit, requiring concentrations greater than 1. mu.g/. mu.L and determining the purity of the plasmids after extraction (A260/280. apprxeq.1.8, A260/230> 2.0).
(2) Cell culture: HEK293T cells were digested with 0.25% trypsin at a cell density of 1X 107Cell/15 cm petri dish, 37 deg.C, 5% CO2The cells were cultured overnight in a cell incubator.
(3) Transfection: when the cell density reaches about 90%, the transfection can be carried out, the fresh DMEM + 10% FBS culture medium is replaced before 2 hours, and plasmid and PEI transfection complexes are prepared, wherein the specifications of the plasmids and PEI transfection complexes are as follows (taking a 10 × 15cm culture dish 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 for 15 minutes, 1mL transfection complex was added to each dish, 5% CO at 37 ℃ C2The cell culture box is cultured for 48-72 hours overnight. After transfection, the cells and the culture medium are collected into a centrifuge tube together, centrifuged, and culture medium supernatant and cell sediment are respectively harvested: PEG8000 precipitation of virus in the culture medium supernatant; cracking the cell precipitate for detoxification; adeno-associated virions obtained from the cell pellet and supernatant were pooled.
(4) Virus purification, titer determination: purifying the virus by using iodixanol ultracentrifugation, determining the virus titer by adopting fluorescent quantitative PCR (polymerase chain reaction), and finally obtaining the recombinant adeno-associated virus titer larger than 1 × 1013viral genome/mL。
(5) Serotype: in this example the recombinant adeno-associated virus was packaged as serotype 2/1.
The HEK293T cell is infected by the double-stranded recombinant adeno-associated virus (scAAV2/1-hSyn-H2B-mclover3) obtained by packaging, a bright field graph shows that the cell growth state is good, and fluorescent protein expression with strong signals can be observed under both a low power lens and a high power lens, and the results are shown in FIG. 2 and indicate that the virus can be normally expressed.
Example 2
Non-specific cell type labeling systems were used to resolve the postsynaptic neuronal cell body distribution throughout the mouse AUD brain.
Marking of living body: the double-stranded recombinant adeno-associated virus (scAAV2/1-hSyn-H2B-mclover3) which is packaged and realizes the cell body labeling of the non-specific cell type postsynaptic neurons is injected into the Auditory cortex (audiocortix, AUD) brain area (AP: -3.0mm, ML: -3.8mm, DV: -1.9mm) of a wild mouse in a volume of 100nL until the virus is expressed for four weeks. Perfusion sampling of the marked sample is respectively used for manual section imaging and whole brain continuous cutting imaging.
Manual slice imaging: rinsing a fixed marked rat brain sample after perfusion with 0.01M PBS, dehydrating the sample in 30% sucrose for 48-72 hours (time is determined according to dehydration conditions, and the judgment is that the rat brain is completely sunk into the bottom), embedding the dehydrated sample by using an OCT frozen section embedding agent, continuously and manually sectioning the sample by using a frozen microtome at the thickness of 50 mu M (the section range is between Bregma +3.0mm and Bregma-7.0 mm), repeatedly staining the brain piece for 10-15 minutes by using 4',6-diamidino-2-phenylindole (4',6-diamidino-2-phenylindole, DAPI) after sequentially pasting the brain piece from front to back, rinsing, sealing the brain piece by using an anti-fluorescence quenching sealing piece agent, and coating nail polish on the periphery of the brain piece for long-term storage. Brain slices containing soma signals were screened under a fluorescent microscope and representative brain slices were imaged.
Whole brain continuous cutting imaging: the perfused marked rat brain samples were embedded using Lowicryl HM20 resin, using the following procedure: rinsing (rat brain in 0.01M PBS 3 times, first two times 6 hours, third overnight) → dehydration (50%, 75%, 95%, 100% and 100% ethanol treatment, each gradient 2 hoursTime) → penetration (successively by 50%, 75%, 100% and 100% resin treatment, first three gradient treatments for 2 hours and last 48 hours) → polymerization (48 ℃ oven standing for 8 hours). Whole brain imaging was performed by a commercial two-color Fluorescence microscopy optical sectioning tomography (fMOST) (model: Biomapping5000) at 0.3X 2 μm3The voxel resolution of (a) is achieved, and a two-color whole brain data containing 4,300 coronal images is acquired after about 4 days of continuous imaging.
And (3) data analysis: the acquired whole brain two-color channel original data is preprocessed, and the maximum value projection (100 μm thickness) is carried out on every 50 coronal plane brain slices of the green channel. Then, the two signals are respectively combined with a single coronal plane brain slice (2 μm in thickness) of a corresponding red channel to obtain a data set of two-color maximum projection for signal display. To better show the advantages of the invention, an AUD whole brain projection data set (disclosed on Allen brain research network) is selectedhttps://connectivity.brain- map.org/(ii) a Numbering: 146858006) and post-processing, and then compared to AUD two-color whole brain data obtained with the tagging system of the present invention. The specific method comprises the following steps: selecting a plurality of bicolor coronal facial brain slices at different positions from the two groups of whole brain data sets respectively, registering the bicolor coronal facial brain slices in an Allen standard brain atlas, and then selecting the same brain area to display the signal distribution of the two groups of data sets.
FIG. 3 is an example of the use of a recombinant adeno-associated virus-mediated non-specific cell type labeling system for slice imaging to resolve the cell bodies of postsynaptic neurons in the whole brain of a mouse AUD. 6 representative brain slices (from Bregma-0.9mm to Bregma-4.0mm) of 50 μm thickness were selected to demonstrate the distribution of AUD in the post-synaptic neuron soma of the cortical and subcortical multiple projected brain regions. The brain regions of these cortex include: orbital area (ORB), Anterior Cingulate Area (ACA), Motor area (MO), olfactory area (ECT), Temporal association cortex (TEa), retrobulbar area (RSP), Visual area (VIS), and olfactory inner area (ENT), and the like. The subcortical brain regions include: caudate nucleus (CP), Basolateral amygdala (BLA), thalamus posterolateral nucleus of the thalamus (LP), Parafascicular nucleus (PF), Medial geniculate complex (MG), and Inferior Colliculus (IC), among others.
FIG. 4 is an example of a whole brain imaging application of a recombinant adeno-associated virus-mediated non-specific cell type labeling system for the resolution of postsynaptic neuronal cell bodies in the whole brain range of a mouse AUD. Whole brain data was obtained using fMOST bicolor channel imaging. Maximum value projection (100 mu m thickness) is carried out on each 50 brain slices in the green channel, and then the maximum value projection is combined with a single coronal plane brain slice (2 mu m thickness) in the corresponding red channel respectively to obtain a data set of two-color maximum value projection. The images in the data set are arranged from front to back in sequence to display the distribution of the marked cell bodies of the brain postsynaptic neurons.
FIG. 5 is a selection of the representative brain slice of FIG. 4 to highlight the effect of the markers in a plurality of sub-brain regions. To better demonstrate the advantages of the present invention, the labeling effect of numerous identical brain regions throughout the brain using the labeling system of the present invention and prior art methods (e.g., using AAV-EF1a-EGFP labeling) were compared. Where panel a is the position of 6 representative brain slices taken, panel b is AUD bicolor whole brain data obtained using the marker system of the present invention obtained using fMOST, and panel c is whole brain data modified from AUD axonal projections using AAV1-EF1a-EGFP markers as disclosed in the Allen brain study (https:// connectivity. brand-map. org/; accession No.: 146858006). As shown in this figure, the postsynaptic somal markers achieved by the present invention have the following advantages over axonal markers: (1) the efficiency of the marking is higher: the soma signals of the markers of the present invention are more widely distributed in many brain areas, such as Primary motor area (MOp), Secondary motor area (MOs), Anterior visual area (VISa), and Anteromedial visual area (VISam) of the cortex, temporal combined cortex Tea, etc.; subcortical numerous brain regions such as the caudate nucleus CP, the postthalamic lateral nucleus LP and the retrothalamic complex (PO) in the Dorsal thalamic lateral group, the lateral geniculate complex dorsalis in the Dorsal thalamic geniculate group (LGd), and several subthalamic regions of the superior colliculus: motion-related intermediate grey-colored layers (SCig), motion-related intermediate white-colored layers (SCiw), and motion-related deep grey-colored layers (SCdg); and Mesencephalon Reticuloeus (MRN). (2) The difference of the regulation and control intensity of the postsynaptic neuron signals of the AUD in each layer in the cortex can be accurately judged: in the cortex, such as Primary sensory cortex (SSp) and visual cortex (VIS), there are more neuron soma in layer 5 than in other layers, indicating that AUD has the greatest regulatory strength on these 5 th cortical neurons; in contrast, the distribution of axons labeled with AAV1-EF1a-EGFP is diffuse and not easy to be analyzed quantitatively, so that the signal intensity distribution of axon fibers from AUD in other cortical layers cannot be judged accurately. (3) Finding a new projection brain area, a new projection type: if the invention is used, a certain number of postsynaptic neuron cell bodies can be clearly observed in the corpus callosum, which indicates that the AUD is in loop connection with the specific type of neurons in the brain area, the regulation and control degree of the brain area is judged according to the number of the cell bodies, and the chemical components of the neurons can be identified by means of immunostaining and the like; whereas the use of AAV1-EF1a-EGFP only labeled nerve fibers that project to the contralateral half-brain via this brain region did not achieve the above objectives.
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 the cell bodies of postsynaptic neurons. Furthermore, the invention combines a whole brain imaging system, can more intuitively display the whole distribution of the cell bodies of the specific type of cells in the specific brain area and the brain area at the whole brain level and the distribution of a plurality of brain areas and sub-brain areas in the whole brain range at the three-dimensional level, and can also display and compare the difference of the regulation strength of different experimental individuals, different brain areas and different cell types in the same brain area on different brain areas or different types of post-synaptic neurons in an output loop in parallel in the same sample.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail 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 realizing cell body labeling of postsynaptic neurons in whole brain and application thereof
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aattgcgcat cccctatcag agagggggag gggaaacagg atgcggcgag gcgcgtgcgc 180
actgccagct tcagcaccgc ggacagtgcc ttcgcccccg cctggcggcg cgcgccaccg 240
ccgcctcagc actgaaggcg cgctgacgtc actcgccggt cccccgcaaa ctccccttcc 300
cggccacctt ggtcgcgtcc gcgccgccgc cggcccagcc ggaccgcacc acgcgaggcg 360
cgagataggg gggcacgggc gcgaccatct gcgctgcggc gccggcgact cagcgctgcc 420
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gaaaggcggc aagaagcgca agcgcagccg caaggagagc tattccatct atgtgtacaa 600
ggttctgaag caggtccacc ctgacaccgg catttcgtcc aaggccatgg gcatcatgaa 660
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tggggagttg gccaagcacg ccgtgtccga gggtactaag gccatcacca agtacaccag 840
cgctaagatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga 900
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tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag 360
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tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac 540
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cggcccagcc ggaccgcacc acgcgaggcg cgagataggg gggcacgggc gcgaccatct 960
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taaggcgcag aagaaaggcg gcaagaagcg caagcgcagc cgcaaggaga gctattccat 1200
ctatgtgtac aaggttctga agcaggtcca ccctgacacc ggcatttcgt ccaaggccat 1260
gggcatcatg aattcgtttg tgaacgacat tttcgagcgc atcgcaggtg aggcttcccg 1320
cctggcgcat tacaacaagc gctcgaccat cacctccagg gagatccaga cggccgtgcg 1380
cctgctgctg cctggggagt tggccaagca cgccgtgtcc gagggtacta aggccatcac 1440
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ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca 1560
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gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcgg 1680
ctacggcgtg gcctgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa 1740
gtccgccatg cccgaaggct acgtccagga gcgcaccatc tctttcaagg acgacggtac 1800
ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct 1860
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caagatccgc cacaacgttg aggacggcag cgtgcagctc gccgaccact accagcagaa 2040
cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gccatcagtc 2100
caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac 2160
cgccgccggg attacacatg gcatggacga gctgtacaag taaagcggcc gctaggcctc 2220
acctgcgatc tcgatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca 2280
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agggggaggt gtgggaggtt ttttaaacta gtccactccc tctctgcgcg ctcgctcgct 2400
cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 2460
gagcgagcga gcgcgcagag agggacagat ccgggcccgc atgcgtcgac aattcactgg 2520
ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg 2580
cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt 2640
cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc 2700
atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg 2760
catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 2820
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atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 3060
tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 3120
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ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 3540
aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 3600
aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa 3660
tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 3720
aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 3780
cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 3840
ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga 3900
gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 3960
agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 4020
atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 4080
cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 4140
cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 4200
cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 4260
tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact 4320
tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 4380
ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 4440
aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 4500
cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 4560
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
gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa g 4911

Claims (10)

1. A recombinant adeno-associated virus, wherein the preparation method of the recombinant adeno-associated virus comprises: the fluorescent protein element of the nuclear localization sequence H2B is coupled through the mediation of a promoter to construct a double-stranded vector, and then the double-stranded vector is packaged into the recombinant adeno-associated virus by a packaging cell.
2. The recombinant adeno-associated virus according to claim 1 wherein the promoter is selected from hSyn, EF1 α, CMV, CAG, CaMKII, Dlx, TH.
3. The recombinant adeno-associated virus 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 thereof.
4. The recombinant adeno-associated virus according to claim 1 wherein the expression of the fluorescent protein element coupled to nuclear localization sequence H2B is expressed alone or mediated by a recombinase recognition sequence, a tetracycline response sequence.
5. The recombinant adeno-associated virus according to claim 1, wherein the recombinase recognition sequence is selected from the group consisting of loxP-STOP-loxP, FLEX, DIO, fDIO, dDIO, vcDIO and scDIO.
6. The recombinant adeno-associated virus according to claim 1 wherein the packaging cell is a HEK293T cell.
7. Use of the recombinant adeno-associated virus according to any one of claims 1 to 6 for neuronal cell body labeling.
8. The use of claim 7, wherein the neuronal cell is a postsynaptic neuronal cell of multiple cell types throughout the brain.
9. The use according to claim 7, wherein the subject is a wild-type rodent, mammalian, or the like, transgenic animal strain expressing a recombinase or tetracycline transcriptional activator, and/or a non-human primate.
10. A labeled preparation of a cell body of a postsynaptic neuron, comprising the recombinant adeno-associated virus according to any one of claims 1 to 6.
CN202111520739.1A 2021-12-13 2021-12-13 Recombinant adeno-associated virus for realizing whole brain postsynaptic neuron cell body marking and application thereof Active CN114107231B (en)

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