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Method for rapidly establishing animal model of Parkinson's disease through substantia nigra gene knockout

A technology for establishing methods and animal models, applied in the biological field, can solve problems such as increasing the difficulty of research, and achieve the effect of stable phenotype

Active Publication Date: 2018-12-14
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, there are many uncertainties and unpredictability in whether the strategy of knocking out the PINK1 gene can successfully produce PD animal models, and the huge differences between different species of animals further increase the difficulty of research
At the same time, there have been no reports about successfully obtaining a large animal model of PD by knocking out this gene. Therefore, the technical bottleneck and technical difficulties need to be overcome; how to construct a PD model that is more similar to humans has also become a PD important topics in disease research

Method used

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  • Method for rapidly establishing animal model of Parkinson's disease through substantia nigra gene knockout
  • Method for rapidly establishing animal model of Parkinson's disease through substantia nigra gene knockout
  • Method for rapidly establishing animal model of Parkinson's disease through substantia nigra gene knockout

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Embodiment 1

[0059] The transformation of embodiment 1 expression vector

[0060] Transform viral vectors expressing target plasmids such as figure 1 , the specific construction process method is as follows (relevant specific operations can be realized by conventional technical means in the field):

[0061] 1. Use the Xba1 and AgeI restriction sites on the pAAV-pMecp2-SpCas9-spA (purchased from Addgene, px551, plasmamid#60957) backbone plasmid to delete the Mecp2 promoter sequence, and connect the Xba1 and AgeI restriction sequences The CMV promoter sequence, so as to realize the expression of SpCas9 in each cell in the brain, and obtain AAV-Cas9.

[0062] 2. For the transformation of the AAV-U6sgRNA-hSyn-GFP (Addgene, px552, plasmamid#60958) plasmid, the hSyn sequence in the original vector was transformed into the CMV promoter sequence through ApaI and BamHI, and cut by BamHI and EcoRI Point to replace the GFP sequence with the RFP sequence to get the AAV-U6sgRNA-CMV-RFP vector.

[00...

Embodiment 2

[0064] Example 2 Constructing the sgRNA vector of CRISPR / Cas9 containing monkey PINK1 gene target sequence

[0065] 1. Design of sgRNA sequence

[0066] In order to screen the PINK1 sgRNA sequence capable of gene editing and with small off-target effects, the applicant used NCBI's Blast software to screen the monkey PINK1 protein coding region gene (SEQ ID NO.1) with low off-target effects and consistent with the CRISPR / Cas9 binding genome Featured (5'-G(19N)-NGG3') or (5'-CCN(19N)-C-3') target sequence sites.

[0067] Inventors in monkey PINK1 gene No. 2 and exon No. 4 ( figure 2 ) respectively designed multiple sgRNAs, namely: PINK1exon2-T1, T2, T3 ( image 3 ) and PINK1exon4-T1, T2, T3 ( Figure 4 ), wherein the PINK1 exon2-T target sequence contains a specific Hpy188I restriction site sequence to facilitate the detection of mutations. The specific sequence information of sgRNA is shown in Table 1:

[0068] Table 1 sgRNA sequence information

[0069]

[0070]

...

Embodiment 3

[0111] Example 3 Virus Packaging and Construction of Animal Models

[0112] The viral vectors AAV-sgRNA-PINK1 Exon2 and AAV-sgRNA-PINK1 Exon4 transformed and constructed in Example 2 and the AAV-Cas9 constructed in Example 1 were used for virus packaging. For specific steps, see the literature Yang et.al., J Clin Invest, 2017 (doi:10.1172 / JCI92087), and injected into the substantia nigra of monkey brain through stereotaxic injection method, the operation method is as follows Figure 9 shown. A total of 11 cynomolgus monkeys were used to construct the animal model, of which 6 were male and 5 were female; 3 were 3-year-old animals, 4 were 8-year-old animals; and 4 were 12-year-old animals.

[0113] The specific injection scheme is as follows: stereotaxic positioning of the substantia nigra of the monkey brain through nuclear magnetic resonance scanning images (MRI), and the parameters are based on the actual measurement of the MRI. Subsequently, 12 μL was injected into the right...

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Abstract

The invention provides a method for rapidly establishing an animal model of Parkinson's disease through substantia nigra gene knockout. The invention discloses sgRNA capable of efficiently specifically knocking out PINK1 genes, and further provides a method for establishing the animal model of Parkinson's disease by virtue of targeted knockout of PINK1 genes in substantia nigra of animals. The method specifically comprises the following steps: injecting sgRNA and CRISPR nuclease into a substantia nigra part of the animal brain, and causing target fragment deletion to the PINK1 genes. Obvious typical dyskinesia symptoms of Parkinson's disease can occur within 3-4 weeks, and the dyskinesia cannot restore without drug therapy. The method directly causes neuronal death of the substantia nigrapart, does not have any side effect, has the mold establishment success rate of 90% or higher, has phenotypic stability and excellent applicability and repeatability, provides important models for screening drugs for the Parkinson's disease, performing stem cell treatment and gene defect repair treatment, researching pathogenesis of deletion of the PINK1 genes and the like, and has huge economic value and preclinical study significances.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for quickly establishing an animal model of Parkinson's disease by knocking out the substantia nigra gene. Background technique [0002] Parkinson's Disease (PD), as the second largest neurodegenerative disease after Alzheimer's disease (AD), has an incidence rate as high as 1-2% among people over 65 years old in China (Pringsheim et al., 2014; Koprich et al. 2017). The clinical symptoms of Parkinson's disease are dyskinesia, resting tremor, muscle stiffness and so on. The pathological changes of the disease are mainly due to the degeneration and necrosis of dopaminergic nerve cells in the substantia nigra of the midbrain and the appearance of Lewy bodies (Dawson et al., 2010). Due to the death of a large number of dopamine neurons, the dopamine in the patient's brain is severely reduced, resulting in the movement disorder of Parkinson's disease. With the incr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N9/22A01K67/027
CPCA01K67/0275A01K2227/10A01K2267/0318C12N9/22C12N15/113C12N15/907C12N2310/10
Inventor 郭祥玉杨伟莉李世华李晓江
Owner JINAN UNIVERSITY
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