CN111778285A - Method for accelerating prion propagation of tau pathology in brain of C57BL6 mouse - Google Patents
Method for accelerating prion propagation of tau pathology in brain of C57BL6 mouse Download PDFInfo
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Abstract
The invention discloses a method for accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse, which comprises the steps of constructing an RNAi adeno-associated virus vector; co-transfecting AAV-293 cells by using a virus-packaged helper plasmid and an RNAi adeno-associated virus vector (high-quality recombinant vector) of the target gene to obtain an RNAi effective vector adeno-associated virus; injecting the virus into a C57BL/6 mouse; constructing a target gene overexpression vector, co-transfecting 293T cells with the target gene overexpression vector and RNAi (ribonucleic acid interference) adeno-associated virus vectors of the target gene, observing cell fluorescence to ensure that the transfection efficiency is more than or equal to 70%, collecting cell extracted protein, and detecting the target gene knockdown condition. The rAAV9 gene can be used as an effective tool carrier for delivering genes in brain tissues, and the speed of prion propagation of tau pathology in mouse brain is accelerated.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a method for accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse.
Background
Alzheimer Disease (AD), commonly known as senile dementia, is a multifactorial disease, with aging being the leading cause. AD possesses typical pathological features: the deposition of Senile Plaques (SPs) by extracellular β -amyloid protein (a β) aggregation and the aggregation of large amounts of tau protein within cells to neurofibrillary tangles (NFTs) and extensive neuronal degeneration. In the field of AD research, a β has been the center of research, and a β has been the target of drug development. Unfortunately, AD is always a serious disaster area for new drug development, the failure rate of clinical tests in the field is as high as 99.6%, and the research in the field is almost completely covered in the past 20 years, so that the search for new targets is very necessary.
The amount of NFTs aggregated and the degree of dementia of the patients were clearly and positively correlated, and were considered as the pathological basis for neuronal fiber degeneration (neurofibrillary degeneration) in AD patients. Abnormal hyperphosphorylation of tau is considered to be an important factor in the aggregation of NFTs and the propagation of tau prions. To further confirm the relationship between tau pathology development, progression and phosphorylation, and to develop AD drugs targeting tau, we sought to knock down the enzyme PP2A that phosphorylates tau and observe changes in tau prion propagation.
PP2A, protein phosphatase 2, is an enzyme encoded by the PPP2CA gene in humans, is a heterotrimer, consisting of a catalytic subunit C, a structural subunit a and a regulatory subunit B. Is a serine threonine phosphorylase, and regulates most phosphorylases in eukaryotic cells, affecting many pathways within the cell.
The recombinant adeno-associated virus rAAV is a gene vector modified on the basis of non-pathogenic wild adeno-associated virus (AAV), has the advantages of extremely low immunogenicity, high safety, wide host cell range, strong diffusion capacity, long in-vivo gene expression time and the like, and is considered to be one of the most promising gene research and gene therapy vectors. The rAAV vector has various types and a plurality of serotypes, and the rAAV vectors of different serotypes can recognize different cell surface receptors and show infection affinities of different tissues or cells.
In recent years, gene therapy has been rapidly developed as a novel therapy which is most drawing attention globally, and rAAV has been a major focus of widespread attention as a star vector for gene therapy. The results of the research show that the rAAV9 can be used as an effective tool vector for delivering genes in brain tissues, researches the molecular mechanism of tau pathology generation through the knock-down of the PP2A gene mediated by a virus vector, defines the relation between tau pathology aggregation and prion propagation and PP2A, prompts the feasibility of gene therapy mediated by the rAAV vector, can also be helpful for screening out drugs for inhibiting or reversing the development and propagation of tau pathology, and provides a new strategy for the treatment of the diseases.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above-mentioned technical drawbacks. In order to determine the relationship between the generation and development of tau pathology and PP2A, a recombinant adeno-associated virus rAAV9-Ppp2ca-RNAi is constructed, and the change of tau pathology is observed by knocking down PP 2A.
Accordingly, in one aspect of the present invention, the present invention overcomes the deficiencies of the prior art by providing a method for accelerating the prion propagation of tau pathology in the brain of C57BL6 mice.
In order to solve the technical problems, the invention provides the following technical scheme: a sequence for accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse, comprising,
target, sequence information is shown as SEQ ID NO 1;
ppp2ca-RNAi-a, sequence information is shown as SEQ ID NO 2;
ppp2ca-RNAi-b, sequence information is shown as SEQ ID NO. 3;
PSC-1, with sequence information as SEQ ID NO 4;
ppp2ca (53604-1) -P1, the sequence information is shown as SEQ ID NO: 5;
ppp2ca (53604-1) -P2, the sequence information is shown as SEQ ID NO: 6;
CMV-F, sequence information as SEQ ID NO 7;
pEGFP-N-3, sequence information is shown as SEQ ID NO 8;
the sequence information is shown as SEQ ID NO 9.
As another aspect of the present invention, the present invention provides a method for accelerating the prion propagation of tau pathology in the brain of C57BL6 mice, comprising, constructing an RNAi adeno-associated viral vector; co-transfecting AAV-293 cells by using a virus-packaged helper plasmid and an RNAi adeno-associated virus vector (high-quality recombinant vector) of the target gene to obtain an RNAi effective vector adeno-associated virus; the virus was injected into C57BL/6 mice.
As a preferred embodiment of the method for accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse, according to the present invention, wherein: constructing a target gene overexpression vector, co-transfecting 293T cells with the target gene overexpression vector and RNAi (ribonucleic acid interference) adeno-associated virus vectors of the target gene, observing cell fluorescence to ensure that the transfection efficiency is more than or equal to 70%, collecting cell extracted protein, and detecting the target gene knockdown condition.
As a preferred embodiment of the method for accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse, according to the present invention, wherein: the constructed RNAi adeno-associated virus vector comprises one or more of Ppp2ca-RNAi-a and Ppp2 ca-RNAi-b.
As a preferred embodiment of the method for accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse, according to the present invention, wherein: the RNAi adeno-associated virus vector is constructed by using a Target sequence and a U6-MCS-CAG-EGFP vector.
As a preferred embodiment of the method for accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse, according to the present invention, wherein: the constructed target gene overexpression vector has the target gene Ppp2ca, the vector name GV143 and the enzyme cutting site XhoI/KpnI.
As a preferred embodiment of the method for accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse, according to the present invention, wherein: the adeno-associated virus of the RNAi effective vector is rAAV9-Ppp2ca-RNAi, and the virus titer can reach 3.56E +12 vg/ml.
As a preferred embodiment of the method for accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse, according to the present invention, wherein: the virus was injected into C57BL/6 mice, i.e., it was injected in the ventricles of the brain.
As a preferred embodiment of the method for accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse, according to the present invention, wherein: the green fluorescence of GFP in the brain of mice can be detected in C57BL/6 mice injected with the virus for 8 months, which suggests that the gene carried by the rAAV9 virus vector can be stably expressed in the brain tissue for a long time.
The invention has the beneficial effects that:
the invention discovers that the rAAV9 gene can be used as an effective tool vector for delivering genes in brain tissues, researches the molecular mechanism of tau pathology generation through the knock-down of a virus vector mediated PP2A gene, confirms the relation between tau pathology aggregation and prion propagation and PP2A, prompts the feasibility of gene therapy mediated by the rAAV9 vector, can also be helpful for screening out drugs for inhibiting or reversing the development and propagation of tau pathology, and provides a new strategy for the treatment of the diseases.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is an image of brain sections 21 days after virus expression, with the arrow indicating the GFP fluorescence signal. A, ICV is injected with rAAV9, B, and hippocampal is injected with rAAV 9. The result shows that the mouse injected with rAAV9 through ICV has a strong fluorescence signal in the right hippocampus and spreads to the left hippocampus, while the virus fluorescence signal injected into the right hippocampus is limited in the hippocampus and does not spread to the contralateral ventricle;
FIG. 2 is a GFP fluorescence signal result chart, wherein after injecting an unloaded virus and injecting a rAAV9-Ppp2ca-RNAi virus, and 8 months after the mouse is infected by the rAAV9 vector, the GFP fluorescence signal can be seen in both the injected unloaded virus and the rAAV9-Ppp2ca-RNAi virus (FIG. 2 and FIG. 3), which indicates that the gene carried by the rAAV9 virus vector can be stably expressed in brain tissue for a long time;
FIG. 3 is a graph showing the results of GFP fluorescence signals: a, the Ipsilateral (IP) brain PP2A expression of mice injected with shPP2A virus in the lateral ventricle of brain is reduced, but the reduction of the contralateral brain PP2A (CON) is not obvious. The PP2A expression was not reduced in mice injected with the empty virus, either in the injection assay or in the contralateral side. b, GFP was detected in the ipsilateral brain injected with shPP2A virus and unloaded virus, and very little GFP was detected in the contralateral side. C57BL/6 mice injected with rAAV9-Ppp2ca-RNAi in the same lateral ventricle have different degrees of pathological changes of tau after 8 months of AD O-tau injection, and have significant difference (p is less than 0.001) compared with a control group injected with unloaded virus;
FIG. 4 is a cell test chart of tau pathology, after the adeno-associated virus vector knockdown PP2A, pathological aggregation of tau was clearly observed in the hippocampus of C57BL/6 mice, fluorescence staining of A and GFP was observed in all four groups of mice, which suggests that genes transmitted by the virus in the mouse brain can be stably expressed, and the aggregation of tau in cells can be seen by immunofluorescence staining of AT8 (indicated by arrows). Counting cells with tau pathology, and statistically analyzing to show that the aggregation and propagation of tau pathology of the AD O-tau-induced C57BL/6 mouse are accelerated by injecting rAAV9-Ppp2 ca-RNAi;
FIG. 5 is a diagram of a Marker strip obtained by the test;
FIG. 6 is a roadmap for the construction of RNAi adeno-associated viral vectors;
FIG. 7 is a schematic diagram showing the construction of an overexpression vector for a target gene;
FIG. 8 is a roadmap for the detection of overexpression plasmid expression;
FIG. 9 is a roadmap for Western Blot exogenous screening of effective RNAi vectors;
FIG. 10 is a roadmap for the adeno-associated viral packaging of an RNAi-effective vector.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example (b):
1: extrasieve adeno-associated virus
1. The RNAi adeno-associated virus vector is constructed by the following route:
siRNA target design
Target sequence information: CTGCGAGAAGGCTAAAGAAAT, GC content (%) 42.86%
Carrier numbering and frame structure
GV478：U6-MCS-CAG-EGFP
RNAi framework
Synthesis of fragments
Ppp2ca-RNAi-a：accggCTGCGAGAAGGCTAAAGAAATttcaagagaATTTCTTTAGCCTTCTCGCAGttttt
Ppp2ca-RNAi-b：tctaaaaaaCTGCGAGAAGGCTAAAGAAATtctcttgaaATTTCTTTAGCCTTCTCGCAGc
Sequencing results
PSC-1
accggCTGCGAGAAGGCTAAAGAAATttcaagagaATTTCTTTAGCCTTCTCGCAGttttt
TGGATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGCTGCGAGAAGGCTAAAGAAATTTCAAGAGAATTTCTTTAGCCTTCTCGCAGTTTTTTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTGCAGCGGCTTCGGAATAGGAACTTCCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTGTTAACGTTAGGGGCGGGATAGCTAGAGCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTCCTCTCACTCTCTGATATTCATTTCTTTGCAAGTTATAAATACTGAATAATAAGATGACATGAACTACTACTGCTAGAGATTTTCCACACTGACTAAAA。
2. Construction of target Gene overexpression vector
(1) Target gene and tool carrier information
The target gene is as follows: ppp2ca
Carrier name: the GV143 is set to a specific position,
enzyme cutting site: XhoI/KpnI
(2) Vector cleavage
Preparing 50 mul enzyme cutting system, gently blowing and beating the system by a pipette, mixing the system evenly, centrifuging the system for a short time, and reacting the system for 3 hours or overnight at 37 ℃. And (4) carrying out agarose gel electrophoresis on the vector enzyme digestion product, and recovering a target band.
| Reagent | Volume (μ l) |
| ddH2O | 42 |
| 10×buffer | 5 |
| Purified plasmid DNA (1. mu.g/. mu.l) | 2 |
| XhoI/KpnI | 1 |
| total | 50 |
(3) Obtaining a target Gene fragment
Amplification primer (containing enzyme cutting protection basic group and enzyme cutting site and containing target gene two-end part sequence for PCR fishing target gene)
PCR amplification of target gene fragment
| Reagent | Volume (μ l) |
| ddH2O | 32.5 |
| |
10 |
| dNTPMix(2.5mM each) | 4 |
| Upstream primer (10. mu.M) | 1 |
| Downstream primer (10. mu.M) | 1 |
| Stencil (10 ng/. mu.l) | 1 |
| PrimeSTAR HS DNA polymerase | 0.5 |
| Total | 50 |
Reaction conditions
(4) Exchange of PCR products with vectors
The following reaction system was prepared in an ice-water bath. And lightly blowing and beating the mixture by using a pipettor, and centrifuging the mixture for a short time to avoid generating bubbles. The reaction was carried out at 37 ℃ for 30min, followed by cooling in an ice-water bath for 5min and then immediately followed by conversion.
Reaction system
(5) Transformation of
Add 10. mu.L of the exchange reaction product to 100. mu.L of competent cells, flick the tube wall and mix well, and leave on ice for 30 min. Heat shock at 42 deg.C for 90s, and incubating in ice water bath for 2 min. Adding 500. mu.L LB medium, and shaking-culturing at 37 deg.C for 1 h. Taking a proper amount of bacterial liquid, uniformly coating the bacterial liquid on a flat plate containing corresponding antibiotics, and carrying out inverted culture in a constant-temperature incubator for 12-16 h.
(6) Colony PCR identification
Preparing the following reaction system, shaking, mixing uniformly, and centrifuging for a short time. Picking single colony in a super clean bench by using a sterile gun head to a 20 mu L identification system, blowing, uniformly mixing, and placing in a PCR instrument for reaction.
Identification of reaction System
| Reagent | Volume (μ L) |
| ddH2O | 9.2 |
| 2xTaq |
10 |
| Upstream primer (10. mu.M) | 0.4 |
| Downstream primer (10. mu.M) | 0.4 |
| | |
| Total | |
| 20 |
Identifying primer (for colony PCR identification of transformant)
| CMV-F | CGCAAATGGGCGGTAGGCGTG |
| pEGFP-N-3 | CGTCGCCGTCCAGCTCGACCAG |
PCR reaction conditions
And (3) analyzing a sequencing result:
TAGTGAACCGTCAGATCCGCTAGCCGCCACCATGGACTACAAGGATGACGATGACAAGGATTACAAAGACGACGATGATAAGGACTATAAGGATGATGACGACAAACCGCTAGCGCTACCGGACTCAGATCTCGAGATGGACGAGAAGTTGTTCACCAAGGAGCTGGACCAGTGGATCGAGCAGCTGAACGAGTGCAAGCAGCTCTCCGAGTCCCAGGTCAAGAGCCTCTGCGAGAAGGCTAAAGAAATCCTGACAAAAGAATCCAACGTTCAAGAGGTTCGATGTCCAGTCACTGTGTGTGGAGATGTACATGGGCAATTTCATGATCTCATGGAACTCTTTAGAATTGGTGGTAAATCACCAGATACAAATTACCTGTTTATGGGAGACTATGTGGACAGAGGATATTACTCTGTTGAAACAGTTACACTGCTTGTAGCTCTTAAGGTTCGTTACCGAGAGCGCATCACCATACTCCGAGGGAATCACGAGAGCAGACAGATCACACAGGTTTATGGGTTCTACGACGAGTGTTTAAGGAAATACGGAAATGCAAATGTTTGGAAATACTTCACAGACCTTTTTGACTATCTTCCTCTCACTGCCTTGGTGGATGGGCAGATCTTCTGTCTACACGGTGGTCTGTCACCATCCATAGACACACTGGATCACATCCGAGCACTCGATCGCCTACAGGAAGTTCCTCATGAGGGTCCAATGTGTGACTTGCTGTGGTCAGATCCAGATGACCGTGGTGGCTGGGGGATATCTCCTCGGGGAGCTGGTTATACCTTTGGCCAAGATATTTCTGAGACATTTAATCATGCCAATGGCCTCACGTTGGTGTCCAGAGCTCACCAGCTGGTGATGGAGGGATATAACTGGTGCCATGACCGGAACGTAGTAACAATTTTCAGTGCTCCAAACTATTGCTATCGTTGTGGTAACCAAGCTGCAATCATGGAACTTGACGACACTCTTAAGTATTCTTTCTTGCAGTTTGACCCAGCACCTCGTAGAGGCGAGCCACATGTCACTCGTCGTACCCCAGACTACTTCCTGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGTCCAGAGCTCACCAGCTGGTGATGGAGGGATATAACTGGTGCCATGACCGGAACGTAGTAACAATTTTCAGTGCTCCAAACTATTGCTATC。
3. overexpression plasmid expression detection
Transfecting 293T cells with a target gene overexpression vector (eukaryotic expression vector), observing cell fluorescence through a fluorescence microscope, collecting cells, extracting proteins, detecting the target gene expression condition through Western blot, and displaying the result as shown in figure 5,
m: marker strip size as shown;
positive 1: WB standard-with 3 × FLAG tag, fusion GFP gene (molecular size: 48 KDa);
con 2: a sample of 293T cells;
OE 3: 293T transfected samples for the plasmids of interest.
The size of the target gene fusion fragment in expression cloning is as follows: 1725 bp;
size of the target gene fusion protein: 63 KDa;
by detecting 293T transfected samples through Western Blot, a characteristic band is observed near 63KDa, and the size of the characteristic band is consistent with that of a target gene fusion protein.
4. Western Blot exogenous screening effective RNAi vector
First 3 interfering plasmids were constructed, 3 were all transfected, then WB verified which was the best, if not verified to be effective, we redesigned 3, then verified, optimized and fumbled.
5. RNAi effective vector adeno-associated virus packaging
Virus titer: rAAV9-Ppp2ca-RNAi 3.56E +12vg/ml
Second, brain ventricle injection AAV virus
Four-month old C57BL/6 mice were anesthetized by intraperitoneal injection of 2.5% 2-Avertin, using a brain stereotaxic apparatus, a microinjection pump and a 31-gauge Hamilton microinjector according to the brain profile of the mice. The constructed rAAV-9 vector is injected into the right ventricle of a mouse by an intracerebroventricular Injection (ICV) mode (the virus titer is 3.56E +12vg/ml) in the following coordinates: bregma is 0.5mm backward, 1.0mm to the left/right side, and 2.5mm from the dura mater to the ventral side. The injection volume is 5 μ l, the injection speed is 0.2 μ l/min, and the needle is left for 10min to prevent the liquid from overflowing.
Thirdly, the separation of AD O-tau (pathological tau extracted from the brain of AD patients):
10% AD brain homogenate was centrifuged at 27,000Xg for 30min at 4 ℃, supernatant (Sup1) was centrifuged at 235,000Xg for 45min at 4 ℃, pellet (Oligo-tau) was washed three times with physiological saline and resuspended in a volume of physiological saline at-80 ℃. This portion of tau is AD O-tau. Salting out, treating with urea, dialyzing, performing ion exchange chromatography, collecting phosphorylated fractions, dialyzing, and storing at-80 deg.C. This portion of tau is AD P-tau.
II, injecting AD O-tau into the hippocampus:
2w after viral injection, 2.5% 2-Avertin was intraperitoneally administered, and the hippocampus was injected with AD P-tau (protein concentration 11mg/ml, amount of tau 600ng/2.5ul) with the following coordinates: bregma was 2.5mm backward, 2.0mm to the left/right side, and 1.8mm needle was inserted from the dura mater to the ventral side. The injection volume is 2.5 μ l, the injection speed is 1.25 μ l/min, and the needle is left for 3min to prevent the liquid from overflowing.
Thirdly, fixing and slicing tissues: taking a mouse, firstly injecting 2.5% of 2-Avertin anesthetic into an abdominal cavity, after anesthesia, opening the chest to expose the heart, inserting a perfusion needle from the apex of the heart to an ascending aorta through a left ventricle, after normal saline is perfused, rapidly perfusing 0.1MPBS (pH7.2) containing 4% paraformaldehyde, after the animal body twitches, regulating the perfusion speed to about 50 drops/min, after the duration is about 30min, taking out the brain, placing the brain into 0.1MPBS (pH7.2) containing 4% paraformaldehyde, and then fixing for 4 h. Then transferring into 20% and 30% sucrose solution, after the tissue block sinks, continuously slicing by freezing coronal section with thickness of 40 μm, and floating in 0.01 MPBS.
Immunohistochemistry
(1) A rinsing sheet with clear hippocampal structure was picked, rinsed 3 times in 0.01M PBS buffer (pH7.2), and then rinsed 3 times in a solution containing 10% triton-100 for 20min and 0.01M PBS buffer (pH 7.2). Placing into a sealing solution containing 10% goat serum, and shaking at room temperature for 2 hr.
(2) The blocked sections were transferred to mouse anti-AT 8 monoclonal antibody diluted AT 1:500, shaken gently for 1h AT room temperature, and then placed in a refrigerator AT 4 ℃ overnight.
(3) The next morning, the primary antibody was aspirated, washed 3 times with PBS, and then 1:500 diluted cy3 fluorescent secondary antibody was added, and hochests (1:500) were protected from light and gently shaken at room temperature for 2 h.
(4)0.01M PBS (pH7.2) was washed 3 times for 10min each in the dark.
(5) And (4) observing under a laser confocal microscope after sealing the fluorescent sealing liquid.
Brain sections were imaged 21 days after virus expression. The results show that the rat brain ventricle tissue injected with rAAV9 by ICV has stronger GFP green fluorescence signal, and the virus injected in the hippocampus spreads to the contralateral side less effectively, so we chose to inject the virus in the lateral ventricle.
The green fluorescence of GFP in the brain of mice can be detected in C57BL/6 mice injected with adeno-associated virus for 8 months, which suggests that the gene carried by the rAAV9 virus vector can be stably expressed in the brain tissue for a long time.
Pathological changes of tau in the brain of mice can be detected in C57BL/6 mice 8 months after injection of rAAV9-Ppp2ca-RNAi, while pathological aggregation of tau does not occur in C57BL/6 mice injected with control virus.
In recent years, gene therapy has been rapidly developed as a novel therapy which is most drawing attention globally, and rAAV has been a major focus of widespread attention as a star vector for gene therapy. The results of the research show that the rAAV9 can be used as an effective tool vector for delivering genes in brain tissues, researches the molecular mechanism of tau pathology generation through the knock-down of the PP2A gene mediated by a virus vector, defines the relation between tau pathology aggregation and prion propagation and PP2A, prompts the feasibility of gene therapy mediated by the rAAV9 vector, can also be helpful for screening out drugs for inhibiting or reversing the development and propagation of the tau pathology, and provides a new strategy for the treatment of the diseases.
The experimental design was divided into four groups of 6-7 mice each, with right ventricle injection of empty-load control virus and right hippocampus injection of physiological saline (con-V + NS), right ventricle injection of empty-load control virus and right hippocampus injection of AD O-tau (con-V + AD O-tau), right ventricle injection of rAAV9-Ppp2ca-RNAi virus and right hippocampus injection of physiological saline (shPP2A + NS), and right ventricle injection of rAAV9-Ppp2ca-RNAi virus and right hippocampus injection of AD O-tau (shPP2A + AD O-tau). Tau pathological aggregation was compared by immunohistochemistry or the like after 8 months of AD O-tau injection.
Viral vector mediated gene level operation has become the mainstream technology of neural research and study; PP2A, protein phosphatase 2, is the major enzyme responsible for dephosphorylation of tau proteins. There are few changes in tau pathology in the normal C57BL/6 mouse brain, and abnormal hyperphosphorylation of tau is thought to be the major factor responsible for tau pathology. In order to better study the influence factors influencing the propagation of tau pathological prion, a recombinant adeno-associated viral vector is constructed, and the influence of tau on pathological changes of tau is observed by knocking down the PP2A level and injecting into a lateral ventricle to reduce the dephosphorylation level of tau. ,
it should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Sequence listing
<110> university of southeast Tong
<120> a method for accelerating the prion propagation of tau pathology in the brain of C57BL6 mice
<141>2020-07-03
<160>9
<170>SIPOSequenceListing 1.0
<210>1
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
ctgcgagaag gctaaagaaa t 21
<210>2
<211>61
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
accggctgcg agaaggctaa agaaatttca agagaatttc tttagccttc tcgcagtttt 60
t 61
<210>3
<211>61
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
tctaaaaaac tgcgagaagg ctaaagaaat tctcttgaaa tttctttagc cttctcgcag 60
c 61
<210>4
<211>732
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
tggattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa 60
taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt 120
accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac 180
accggctgcg agaaggctaa agaaatttca agagaatttc tttagccttc tcgcagtttt 240
ttagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 300
tcggtgcttt ttttctgcag cggcttcgga ataggaactt cccagacatg ataagataca 360
ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa 420
tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttgttaacg 480
ttaggggcgg gatagctaga gccagacatg ataagataca ttgatgagtt tggacaaacc 540
acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 600
tttgtaacca ttataagctg caataaacaa gttcctctca ctctctgata ttcatttctt 660
tgcaagttat aaatactgaa taataagatg acatgaacta ctactgctag agattttcca 720
cactgactaa aa 732
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taccggactc agatctcgag atggacgaga agttgttcac 40
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<213> Artificial Sequence (Artificial Sequence)
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gatcccgggc ccgcggtacc gtcaggaagt agtctggggt ac 42
<210>7
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<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
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cgcaaatggg cggtaggcgt g21
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cgtcgccgtc cagctcgacc ag 22
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<400>9
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ttacaaagac gacgatgata aggactataa ggatgatgac gacaaaccgc tagcgctacc 120
ggactcagat ctcgagatgg acgagaagtt gttcaccaag gagctggacc agtggatcga 180
gcagctgaac gagtgcaagc agctctccga gtcccaggtc aagagcctct gcgagaaggc 240
taaagaaatc ctgacaaaag aatccaacgt tcaagaggtt cgatgtccag tcactgtgtg 300
tggagatgta catgggcaat ttcatgatct catggaactc tttagaattg gtggtaaatc 360
accagataca aattacctgt ttatgggaga ctatgtggac agaggatatt actctgttga 420
aacagttaca ctgcttgtag ctcttaaggt tcgttaccga gagcgcatca ccatactccg 480
agggaatcac gagagcagac agatcacaca ggtttatggg ttctacgacg agtgtttaag 540
gaaatacgga aatgcaaatg tttggaaata cttcacagac ctttttgact atcttcctct 600
cactgccttg gtggatgggc agatcttctg tctacacggt ggtctgtcac catccataga 660
cacactggat cacatccgag cactcgatcg cctacaggaa gttcctcatg agggtccaat 720
gtgtgacttg ctgtggtcag atccagatga ccgtggtggc tgggggatat ctcctcgggg 780
agctggttat acctttggcc aagatatttc tgagacattt aatcatgcca atggcctcac 840
gttggtgtcc agagctcacc agctggtgat ggagggatat aactggtgcc atgaccggaa 900
cgtagtaaca attttcagtg ctccaaacta ttgctatcgt tgtggtaacc aagctgcaat 960
catggaactt gacgacactc ttaagtattc tttcttgcag tttgacccag cacctcgtag 1020
aggcgagcca catgtcactc gtcgtacccc agactacttc ctgacggtac cgcgggcccg 1080
ggatccaccg gtcgccacca tggtgagcaa gtccagagct caccagctgg tgatggaggg 1140
atataactgg tgccatgacc ggaacgtagt aacaattttc agtgctccaa actattgcta 1200
tc 1202
Claims (9)
1. A sequence that accelerates the prion propagation of tau pathology in the brain of C57BL6 mice, comprising: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
target, sequence information is shown as SEQ ID NO 1;
ppp2ca-RNAi-a, sequence information is shown as SEQ ID NO 2;
ppp2ca-RNAi-b, sequence information is shown as SEQ ID NO. 3;
PSC-1, with sequence information as SEQ ID NO 4;
ppp2ca (53604-1) -P1, the sequence information is shown as SEQ ID NO: 5;
ppp2ca (53604-1) -P2, the sequence information is shown as SEQ ID NO: 6;
CMV-F, sequence information as SEQ ID NO 7;
pEGFP-N-3, sequence information is shown as SEQ ID NO 8;
the sequence information is shown as SEQ ID NO 9.
2. A method of accelerating prion propagation in the brain of a C57BL6 mouse of tau pathology, comprising: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
constructing an RNAi adeno-associated virus vector;
co-transfecting AAV-293 cells by using a virus-packaged helper plasmid and an RNAi adeno-associated virus vector (high-quality recombinant vector) of the target gene to obtain an RNAi effective vector adeno-associated virus;
the virus was injected into C57BL/6 mice.
3. The method of accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse, according to claim 2, wherein: constructing a target gene overexpression vector, co-transfecting 293T cells with the target gene overexpression vector and RNAi (ribonucleic acid interference) adeno-associated virus vectors of the target gene, observing cell fluorescence to ensure that the transfection efficiency is more than or equal to 70%, collecting cell extracted protein, and detecting the target gene knockdown condition.
4. The method of accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse, according to claim 2 or 3, wherein: the constructed RNAi adeno-associated virus vector comprises one or more of Ppp2ca-RNAi-a and Ppp2 ca-RNAi-b.
5. The method of accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse, according to claim 4, wherein: the RNAi adeno-associated virus vector is constructed by using a Target sequence and a U6-MCS-CAG-EGFP vector.
6. The method of accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse, according to any one of claims 2, 3 or 5, wherein: the constructed target gene overexpression vector has the target gene Ppp2ca, the vector name GV143 and the enzyme cutting site XhoI/KpnI.
7. The method of accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse, according to any one of claims 2, 3 or 5, wherein: the adeno-associated virus of the RNAi effective vector is rAAV9-Ppp2ca-RNAi, and the virus titer can reach 3.56E +12 vg/ml.
8. The method of accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse, according to any one of claims 2, 3 or 5, wherein: the virus was injected into C57BL/6 mice, i.e., it was injected in the ventricles of the brain.
9. The method of accelerating the prion propagation of tau pathology in the brain of a C57BL6 mouse, according to any one of claims 2, 3 or 5, wherein: the green fluorescence of GFP in the brain of mice can be detected in C57BL/6 mice injected with the virus for 8 months, which suggests that the gene carried by the rAAV9 virus vector can be stably expressed in the brain tissue for a long time.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113430230A (en) * | 2021-06-24 | 2021-09-24 | 南通大学 | Application of tau truncation protein in inducing tau pathological aggregation |
| CN113462722A (en) * | 2021-06-24 | 2021-10-01 | 南通大学 | Method for inhibiting tau pathology prion propagation through adeno-associated virus mediation |
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