A recombinant adeno-associated virus composition and its application in precise sparse labeling

Abstract

The present invention discloses a recombinant adeno-associated virus composition and its application in precise sparse labeling. The adeno-associated virus composition comprises rAAV13 and rAAV9. The rAAV13 expresses Cre recombinase, which has the characteristic of limited infection. The rAAV9 expresses a fluorescent reporter gene dependent on the Cre recombinase. In vivo tests show that through the dual virus strategy, rAAV13 can be combined with rAAV9 after different multiple dilutions to achieve precise sparse highlight labeling. The present invention uses rAAV13 for precise sparse labeling for the first time, providing new tools and technical support for neuroscience research, and has broad application value and market prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a recombinant adeno-associated virus composition and an application thereof in precise sparse labeling. Background Art

[0002] A major feature of the nervous system is the presence of a large number of neurons with different morphological structures and physiological properties. Different types of neurons play different roles in neural circuits, and they are integrated into various neural circuits to perform complex behaviors. The mapping of brain connectivity maps will depict the connections between different brain regions and between neuronal groups in different locations in the brain, but due to the lack of single-cell precision, it cannot accurately reflect the fine structure of the nervous system. Therefore, clarifying the axonal and dendritic projection patterns of individual neurons has become the key to exploring the structure of brain neural circuits.

[0003] Relying on sparse labeling technology to reconstruct individual neurons can not only help us understand their morphological structure in neural circuits, but also help to accurately analyze how neural signals are organized and how they are transmitted to target areas through the brain. Sparse labeling technology is to brightly label individual neurons and accurately analyze the morphological structure of individual neurons in neural circuits, so as to analyze brain structure at the single neuron level and draw single neuron projection maps. Therefore, this technology can not only reconstruct the projection morphology of single neurons throughout the brain, but also analyze the input and output sites of single neuron information, providing support for understanding how single neurons form complex and changeable neural circuits. Although it has been reported that some combinations of viruses can achieve sparse labeling, there is still the problem of a large range of viral diffusion, resulting in unstable or inaccurate spatial distribution. Therefore, it is necessary to develop a combination of viruses with a small diffusion range to achieve accurate sparse labeling of neurons. Summary of the invention

[0004] In order to address the deficiencies in the prior art, the present invention aims to provide a recombinant adeno-associated virus composition and its application in precise sparse labeling.

[0005] The specific technical solutions of the present invention are as follows:

[0006] The present invention provides a recombinant adeno-associated virus composition, the composition comprising a serotype 13 recombinant adeno-associated virus and a serotype 9 recombinant adeno-associated virus;

[0007] The serotype 13 recombinant adeno-associated virus is obtained by packaging a packaging plasmid containing adeno-associated virus type 2 Rep gene and adeno-associated virus type 13 Cap gene, and carries Cre recombinase;

[0008] The serotype 9 recombinant adeno-associated virus is a serotype 9 adeno-associated virus vector that expresses a fluorescent reporter gene and is induced by Cre recombinase. The serotype 9 recombinant adeno-associated virus is obtained by packaging a packaging plasmid containing adeno-associated virus type 2 Rep gene and adeno-associated virus type 9 Cap gene.

[0009] Furthermore, the preparation method of the serotype 13 recombinant adeno-associated virus is as follows: co-transfecting the recombinant adeno-associated virus packaging plasmid, the adeno-associated virus core plasmid and the adenovirus element helper plasmid into a packaging cell line, and then harvesting, purifying and concentrating the virus to obtain;

[0010] Among them, the recombinant adeno-associated virus packaging plasmid carries the Rep gene of type 2 adeno-associated virus and the Cap gene of type 13 adeno-associated virus; the recombinant adeno-associated virus core plasmid contains inverted terminal repeat sequences ITR at both ends, and a promoter, an exogenous gene, a transcription regulatory element and a transcription termination sequence are inserted in the middle of the ITR in sequence, and the exogenous gene includes an SV40 NLS sequence and a Cre gene.

[0011] Preferably, the promoter is hSyn promoter;

[0012] The transcriptional regulatory element is WPRE;

[0013] The transcription termination sequence is SV40 polyA or hGH polyA.

[0014] Furthermore, the preparation method of the serotype 9 recombinant adeno-associated virus is as follows: co-transfecting the recombinant adeno-associated virus packaging plasmid, the adeno-associated virus core plasmid and the adenovirus element helper plasmid into a packaging cell line, and then purifying and concentrating the virus to obtain;

[0015] Among them, the recombinant adeno-associated virus packaging plasmid carries the Rep gene of type 2 adeno-associated virus and the Cap gene of type 9 adeno-associated virus; the recombinant adeno-associated virus core plasmid contains inverted terminal repeat sequences ITR at both ends, and a promoter, a DIO element, an exogenous gene, a transcription regulatory element and a transcription termination sequence are inserted in the middle of the ITR in sequence, and the exogenous gene includes a fluorescent reporter gene.

[0016] Preferably, the promoter is a CAG promoter;

[0017] The fluorescent reporter gene is EGFP;

[0018] The transcriptional regulatory element is WPRE;

[0019] The transcription termination sequence is SV40 polyA or hGH polyA.

[0020] The present invention also provides the use of the recombinant adeno-associated virus composition in preparing precise sparse markers of neurons.

[0021] The present invention further provides a method for precise sparse labeling of neurons, wherein the serotype 13 recombinant adeno-associated virus is diluted, mixed with the serotype 9 recombinant adeno-associated virus at a volume ratio of 1:1, and then injected into the target brain area.

[0022] Furthermore, the serotype 13 recombinant adeno-associated virus is diluted 1000-10000 times.

[0023] The beneficial effects of the present invention are:

[0024] The present invention further combines the rAAV13 vector, which has the precise labeling characteristics of small-scale diffusion, with the rAAV9 vector for precise sparse labeling of neurons. In vivo tests have shown that through the dual-virus strategy, rAAV13 can be combined with rAAV9 after different multiple dilutions to achieve precise sparse highlight labeling. The present invention applies the rAAV13 vector to the viral sparse labeling strategy for the first time, achieving sparse highlight labeling of neurons in a small range and precise sites, providing new tools and technical support for neuroscience research, and has broad application value and market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a map of the recombinant adeno-associated virus serotype packaging plasmid pAAV-RC2 / 13 expression vector.

[0026] Figure 2 The rAAV2 / 13-hSyn-SV40 NLS-Cre-WPRE-hGH polyA virus was diluted 1000 times and mixed with the rAAV2 / 9-CAG-DIO-EGFP-WPRE-hGH polyA virus to label the signal in the S1 brain region of C57BL / 6J mice.

[0027] Figure 3 The rAAV2 / 13-hSyn-SV40 NLS-Cre-WPRE-hGH polyA virus was diluted 10,000 times and mixed with the rAAV2 / 9-CAG-DIO-EGFP-WPRE-hGH polyA virus to label the signal in the S1 brain region of C57BL / 6J mice. DETAILED DESCRIPTION

[0028] In order to understand the present invention more clearly, the present invention is further described with reference to the following examples and accompanying drawings. The examples are only used for explanation and are not intended to limit the present invention in any way. In the examples, each raw material reagent is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0029] Example 1

[0030] 1. Preparation of recombinant adeno-associated virus

[0031] (1) Preparation of rAAV2 / 13-hSyn-SV40 NLS-Cre-WPRE-hGH polyA virus

[0032] According to the AAV13 genome sequence (GenBank: EU285562), the AAV13 Cap gene sequence was synthesized and used as a template. The AAV13 Cap gene fragment was amplified using Takara Primerstar Polymerase (Takara). The sequence of the forward primer Cap13-F is shown in SEQ ID NO.1, and the sequence of the reverse primer Cap13-R is shown in SEQ ID NO.2. The amplified DNA fragment was recovered using a gel recovery kit (Omega).

[0033] The pAAV-RC2 / 1 vector (purchased from Addgene, #112862) and the AAV13 Cap gene fragment were digested with restriction endonucleases SwaI and AgeI (New England Biolabs), respectively. The AAV13 Cap gene fragment was then inserted into pAAV-RC2 / 1 using T4 ligase. The ligation product was transformed into competent Stbl3. The clones identified as positive by colony PCR were inoculated into 15 ml of LB liquid medium for culture and plasmids were extracted for sequencing. The clones with correct sequencing were named pAAV-RC2 / 13, and the plasmid obtained was able to encode the AAV13 capsid protein VP1. The map of the constructed recombinant adeno-associated virus serotype packaging plasmid pAAV-RC2 / 13 expression vector is shown in the figure. Figure 1 As shown, its gene sequence is shown in SEQ ID NO.3.

[0034] The recombinant adeno-associated virus (AAV) serotype packaging plasmid pAAV-RC2 / 13 expression vector was used to package the recombinant adeno-associated virus. The core element plasmid pAAV-hSyn-SV40 NLS-Cre-WPRE-hGH polyA (Brinkas (Shenzhen) Biotechnology Co., Ltd.) was co-transfected into HEK-293T cells with the pAAV-RC2 / 13 serotype AAV capsid plasmid and the adenovirus element auxiliary plasmid pAdDeltaF6 (Addgene, #112867) at a ratio of 1:1:1. The supernatant and cell pellet were collected 72 hours after transfection, concentrated and purified by iodixanol gradient centrifugation, and the recombinant AAV titer was detected by SYBR Green qPCR. The titer of the rAAV2 / 13-hSyn-SV40 NLS-Cre-WPRE-hGH polyA virus was 4×10 13 VG / mL.

[0035] (2) Preparation of rAAV2 / 9-CAG-DIO-EGFP-WPRE-hGH polyA virus

[0036] The serotype plasmid pAAV2 / 9n was purchased from Addgene (#112865). The recombinant adeno-associated virus serotype packaging plasmid pAAV2 / 9n expression vector was used to package the recombinant adeno-associated virus. The core element plasmid pAAV-CAG-DIO-EGFP-WPRE-hGH polyA (Brinkas (Shenzhen) Biotechnology Co., Ltd.) was co-transfected into HEK-293T cells with the pAAV2 / 9n serotype AAV capsid plasmid and the adenovirus element auxiliary plasmid pAdDeltaF6 at a ratio of 1:1:1. After 72 hours of transfection, the supernatant and cell pellet were collected, concentrated and purified by iodixanol gradient centrifugation. Finally, the recombinant adeno-associated virus titer was detected by SYBR Green qPCR. The titer of the rAAV2 / 9-CAG-DIO-EGFP-WPRE-hGH polyA virus was 8×10 12 VG / mL.

[0037] II. In vivo application of recombinant adeno-associated virus

[0038] (1) The prepared rAAV2 / 13-hSyn-SV40 NLS-Cre-WPRE-hGH polyA virus was diluted 1000 times and mixed with rAAV2 / 9-CAG-DIO-EGFP-WPRE-hGH polyA virus at a volume ratio of 1:1 (200 nL / mouse). The mixture was injected into the primary somatosensory cortex area (S1) of 8-10 week old C57BL / 6 mice (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.) by brain stereotaxic. The brain was perfused and removed 3 weeks later. The mouse brain tissue was fixed with DEPC-treated PFA solution for 4 hours and then dehydrated with DEPC-treated 30% sucrose-PBS solution for 48 hours. The dehydrated brain tissue was fully embedded with tissue embedding agent and cut into 40 μm thick brain slices with a freezing slicer. The slices were mounted and imaged using a slide scanner microscope. The results of in vivo detection show that the dual virus sparse highlight labeling strategy can accurately and sparsely label the fine structural morphology of neurons. Figure 2 The rAAV2 / 13-hSyn-SV40 NLS-Cre-WPRE-hGH polyA virus was diluted 1000 times and mixed with the rAAV2 / 9-CAG-DIO-EGFP-WPRE-hGH polyA virus to label the signal in the S1 brain region of C57BL / 6J mice.

[0039] (2) The prepared rAAV2 / 13-hSyn-SV40 NLS-Cre-WPRE-hGH polyA virus was diluted 10,000 times and mixed with rAAV2 / 9-CAG-DIO-EGFP-WPRE-hGH polyA virus at a volume ratio of 1:1 (200 nL / mouse). The mixture was injected into the primary somatosensory cortex area (S1) of 8-10 week old C57BL / 6 mice (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.) by stereotaxic brain localization. The brain was perfused and removed 3 weeks later. The mouse brain tissue was fixed with DEPC-treated PFA solution for 4 hours and then dehydrated with DEPC-treated 30% sucrose-PBS solution for 48 hours. The dehydrated brain tissue was fully embedded with tissue embedding agent and cut into 40 μm thick brain slices with a freezing slicer. The slices were mounted and imaged using a slide scanner microscope. The results of in vivo detection show that the dual-virus sparse highlight labeling strategy can accurately and sparsely label the fine structural morphology of neurons. Figure 3 The rAAV2 / 13-hSyn-SV40 NLS-Cre-WPRE-hGH polyA virus was diluted 10,000 times and mixed with the rAAV2 / 9-CAG-DIO-EGFP-WPRE-hGH polyA virus to label the signal in the S1 brain region of C57BL / 6J mice.

[0040] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A recombinant adeno-associated virus composition, It is characterized in that The composition comprises a serotype 13 recombinant adeno-associated virus and a serotype 9 recombinant adeno-associated virus; The serotype 13 recombinant adeno-associated virus is obtained by packaging a packaging plasmid containing adeno-associated virus type 2 Rep gene and adeno-associated virus type 13 Cap gene, and carries Cre recombinase; The serotype 9 recombinant adeno-associated virus is a serotype 9 adeno-associated virus vector that expresses a fluorescent reporter gene and is induced by Cre recombinase. The serotype 9 recombinant adeno-associated virus is obtained by packaging a packaging plasmid containing adeno-associated virus type 2 Rep gene and adeno-associated virus type 9 Cap gene.

2. The recombinant adeno-associated virus composition according to claim 1, It is characterized in that The preparation method of the serotype 13 recombinant adeno-associated virus is as follows: co-transfecting the recombinant adeno-associated virus packaging plasmid, the adeno-associated virus core plasmid and the adenovirus element helper plasmid into the packaging cell line, and then harvesting, purifying and concentrating the virus to obtain; Among them, the recombinant adeno-associated virus packaging plasmid carries the Rep gene of type 2 adeno-associated virus and the Cap gene of type 13 adeno-associated virus; the recombinant adeno-associated virus core plasmid contains inverted terminal repeat sequences ITR at both ends, and a promoter, an exogenous gene, a transcription regulatory element and a transcription termination sequence are inserted in the middle of the ITR in sequence, and the exogenous gene includes an SV40 NLS sequence and a Cre gene.

3. The recombinant adeno-associated virus composition according to claim 2, It is characterized in that The promoter is hSyn promoter; The transcriptional regulatory element is WPRE; The transcription termination sequence is SV40 polyA or hGH polyA.

4. The recombinant adeno-associated virus composition according to claim 1, It is characterized in that The method for preparing the serotype 9 recombinant adeno-associated virus is as follows: co-transfecting the recombinant adeno-associated virus packaging plasmid, the adeno-associated virus core plasmid and the adenovirus element helper plasmid into the packaging cell line, and then purifying and concentrating the virus to obtain; Among them, the recombinant adeno-associated virus packaging plasmid carries the Rep gene of type 2 adeno-associated virus and the Cap gene of type 9 adeno-associated virus; the recombinant adeno-associated virus core plasmid contains inverted terminal repeat sequences ITR at both ends, and a promoter, a DIO element, an exogenous gene, a transcription regulatory element and a transcription termination sequence are inserted in the middle of the ITR in sequence, and the exogenous gene includes a fluorescent reporter gene.

5. The recombinant adeno-associated virus composition according to claim 4, It is characterized in that The promoter is a CAG promoter; The fluorescent reporter gene is EGFP; The transcriptional regulatory element is WPRE; The transcription termination sequence is SV40 polyA or hGH polyA.

6. Use of the recombinant adeno-associated virus composition according to claim 1 in preparing precise sparse markers of neurons.

7. A method for accurate sparse labeling of neurons. It is characterized in that The serotype 13 recombinant adeno-associated virus of claim 1 is diluted 1000-10000 times, mixed with the serotype 9 recombinant adeno-associated virus at a volume ratio of 1:1, and then injected into the target brain area; the titer of the serotype 13 recombinant adeno-associated virus is 4×10 13 VG / mL, and the titer of the serotype 9 recombinant adeno-associated virus was 8×10 12 VG / mL.

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