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AD cell model established based on CRISPR/Cas9 gene editing technology as well as construction method and application of AD cell model

A cell model and gene editing technology, applied in the field of AD cell model, can solve the problem of the relationship between the ADAM10 gene and neurons, etc.

Pending Publication Date: 2021-06-04
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no related reports on the relationship between ADAM10 gene and neurons at this stage

Method used

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  • AD cell model established based on CRISPR/Cas9 gene editing technology as well as construction method and application of AD cell model
  • AD cell model established based on CRISPR/Cas9 gene editing technology as well as construction method and application of AD cell model
  • AD cell model established based on CRISPR/Cas9 gene editing technology as well as construction method and application of AD cell model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] (1) Construction of CRISPR / CAS9 gene editing method

[0079] The following processes are all operated under sterile conditions in the aseptic operating table:

[0080] ①Construction of RNP complex: Take 0.33 μl of crRNA1 (100 μM), 0.33 μl of crRNA2 (100 μM), 0.33 μl of crRNA3 (100 μM) and 1 μl of Alt-R.CRISPR-Cas9 tracrRNA (100 μM) and mix well and use without Dilute the nuclease buffer to a concentration of 20-80 μM to obtain the RNA mixture. The RNA mixture was heated at 95°C for 5 min and cooled at room temperature for 10 min to obtain an annealed RNA mixture. Take 2.9 μl of the annealed RNA mixture and 1 μl of Cas 9 protein according to the ratio of 1.2:1-2:1, mix thoroughly and leave at room temperature for 10 minutes. After adding 0.6 μl electroporation enhancement solution, react at room temperature for 5 minutes to obtain RNP complex.

[0081] ② Electrotransfer of the RNP complex into the SH-SY5Y cells to be knocked out: preheat the medium containing 15% FBS,...

Embodiment 2

[0111] Example 2 Determination of ADAM10 target protein in AD cell model

[0112] The protein level of ADAM10 in the knockout group was verified by Western staining. like Figure 6 As shown in A, compared with the blank control KO group, the expression of ADAM10 protein in the ADAM10 KO group was extremely significantly decreased, which was consistent with the results of Sanger sequencing. The data further indicated that the ADAM10 gene was successfully knocked out in SH-SY5Y cells using CRISPR / Cas 9 gene editing technology. Subsequent experimental research can be carried out.

Embodiment 3

[0113] Example 3 Determination of Aβ and Tau Protein in AD Cell Model

[0114] The accumulation of Aβ and intracellular hyperphosphorylated Tau protein is the main neuropathological criterion for diagnosing AD today, and the ratio of Aβ42 / Aβ40 in the plasma of AD patients can be used as a pre-screening index for AD clinically. Therefore, we verified Aβ; Tau; S-199Tau; S-214Tau:

[0115] Take the cells and discard the culture medium, add an appropriate amount of PBS solution to rinse the cells and discard the solution, add an appropriate amount of Trpsin to digest the cells for several minutes, then add 15% FBS medium to stop the digestion, and centrifuge at 1100rpm for 10min. After the PBS solution is fully resuspended, take a small amount of cells and count them. Take 2,000,000 cells and strictly follow the ELISA kit instructions. The total protein of each sample is determined by the BCA kit. After correcting the total protein, measure Aβ40; The content of Aβ42; p-Tau and Ta...

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Abstract

The invention discloses an AD cell model established based on a CRISPR / Cas 9 gene editing technology as well as a construction method and application of the AD cell model. According to the Alzheimer's disease cell model, an RNP compound composed of crRNA, TracrRNA and Cas9 protein of a targeted ADAM10 gene is electrically transferred into an SH-SY5Y cell, and the target gene ADAM10 is successfully knocked out in the SH-SY5Y cell strain. Compared with normal cells, the cell model disclosed by the invention has the advantages that the expressions of A beta 42, A beta 42 / A beta 40 and pTau / Tau are remarkably improved, the inflammasome NLRP3 protein is remarkably up-regulated, and the expression of a proinflammatory factor TNF-alpha is remarkably improved. Meanwhile, the neural differentiation function of cells is weakened, the length of neurons is shrunk, the growth speed of the cells is remarkably slowed down, the connection of the neurons is remarkably reduced, and the neuronal necrosis disease in the brain of an AD patient can be simulated.

Description

technical field [0001] The invention belongs to the field of biomedicine, relates to an AD cell model established based on CRISPR / Cas 9 gene editing technology and its construction method and application, in particular to using CRISPR / Cas 9 gene editing technology to knock out the ADAM10 gene in SH-SY5Y cells to establish an anti A more comprehensive AD cellular model of neuronal necrosis. Background technique [0002] At present, the number of AD patients in the world exceeds 50 million, which has a huge impact on people's quality of life and health. AD is rated as one of the most devastating diseases due to the high costs incurred in care and management of AD patients [1-3] . Despite decades of research and drug development efforts by scientists on the disease, there is still no drug that can slow the progression of AD [4] . [0003] AD is a genetically heterogeneous disease with complex pathobiology. Accumulation of Aβ and intracellular hyperphosphorylated Tau remain...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N9/22C12N5/10C12Q1/02
CPCC12N15/1137C12N9/22C12N9/6478C12N5/0693C12N5/0618G01N33/5058G01N33/5008C12N2310/20C12N2510/00C12N2503/02G01N2500/10
Inventor 王广基李昔诺阿基业朱哲英徐进宜孙渊
Owner CHINA PHARM UNIV
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