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Method for preparing inhibitory interneurons capable of retaining age characteristics from non-nerve cell transformation

An interneuron, nerve cell technology, applied in nervous system cells, biochemical equipment and methods, animal cells, etc.

Pending Publication Date: 2022-04-01
宁波易赛腾生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite the great demand, there are still no related technologies and products on the market that can produce inhibitory interneurons from normal people or patients at the disease stage with high efficiency and high purity

Method used

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  • Method for preparing inhibitory interneurons capable of retaining age characteristics from non-nerve cell transformation
  • Method for preparing inhibitory interneurons capable of retaining age characteristics from non-nerve cell transformation
  • Method for preparing inhibitory interneurons capable of retaining age characteristics from non-nerve cell transformation

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Experimental program
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preparation example Construction

[0060] For the above-mentioned preparation method, the following steps can be specifically included in the actual preparation application:

[0061]a. Construct the above genes into commercially available or self-owned retroviral vectors, lentiviral vectors or AAV viral vectors, and the promoters regulating the expression level in each vector can be CMV, CAG, EF1α, PGK, TRE Tight, Any one or more of TRE3G. At the same time, the above-mentioned genes can be connected by 2A sequences (for example, T2A, E2A, P2A, F2A, etc.) or IRES sequences from different sources, and a green or red fluorescent reporter gene can be optionally further introduced to facilitate the determination of viral packaging quality and titer. Degree, observation of changes in cell morphology, determination of cell purity and subsequent analysis of various specific applications.

[0062] b. Through cell transfection, the above-mentioned gene vectors are packaged into corresponding retroviruses, lentiviruses o...

Embodiment 1

[0089] Example 1 Rapid and efficient preparation of high-purity inhibitory interneurons from human skin fibroblasts

[0090] 1. Materials

[0091] 1) Cells: 293T cells used for virus packaging were purchased from ATCC (CRL-3216) in the United States; human skin fibroblasts were purchased from Sciencell (product number #2320) in the United States. 293T and human skin fibroblasts were cultured with high-glucose DMEM medium containing 10-20% FBS and 1×P / S double antibody.

[0092] 2) Instruments and reagents:

[0093] A. CO2 cell incubator (ESCO CLM-170B-8-CN); ultra-clean bench (ESCO AC2-5S1); fluorescence microscope (Thermo EVOS M5000); ultra-low temperature refrigerator (Haier DW-86L388J); liquid nitrogen tank (Haier YDS-175-216-F); high-speed refrigerated centrifuge (Baiyang BY-R20 type); normal temperature high-speed centrifuge (Xiangyi H1650-W);

[0094] B. Intelligent high-pressure steam sterilizer (Shanghai Shenan LDZM-80KCS); American SHELLAB drying oven (CE3F-2); ele...

Embodiment 2

[0107] Example 2 Rapid and efficient preparation of high-purity inhibitory interneurons from human embryonic lung fibroblasts

[0108] 1. Materials

[0109] Human embryonic lung fibroblasts MRC-5 were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (ZQ0006). All the other materials are with embodiment 1 (omitted).

[0110] 2. Preparation method

[0111] 1) Plasmid construction: ASCL1, DLX1, LHX6 and SOX4 genes were respectively constructed into retroviral vectors, and CMV was used as the promoter regulating gene expression level in the vectors. At the same time, a green fluorescent reporter gene is introduced through the T2A sequence, so as to determine the quality and titer of virus packaging, observe changes in cell morphology, determine cell purity, and be used in various subsequent specific applications.

[0112] 2) Virus packaging: each plasmid carrying the above-mentioned genes, pGP and pVSV-G was mixed with the transfection reagent Lipofectamine2000...

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Abstract

The invention relates to a method for preparing inhibitory interneurons capable of retaining age and pathological characteristics in the morbidity stage through non-nerve cell transformation. According to the preparation method disclosed by the invention, a key cell direct transdifferentiation gene combination and a promoter for regulating and controlling the expression level are optimized, viruses capable of efficiently infecting donor cells of various ages can be packaged, and an appropriate coating matrix is adopted to promote the donor cells and transformed nerve cells to adhere to the wall to grow; an induced differentiation culture solution containing a small molecule compound capable of promoting transdifferentiation and growth factors is utilized, a large number of inhibitory interneurons are obtained through transdifferentiation after the differentiation culture solution is replaced several times within 10-14 days, and finally the high-purity inhibitory interneurons are obtained through separation and purification.

Description

technical field [0001] The present invention relates to the fields of research and treatment of epilepsy, depression, and schizophrenia, and more specifically, the present invention relates to a related method for preparing inhibitory interneurons that retain the age of onset and pathological characteristics from non-nerve cells . Background technique [0002] Epilepsy (epilepsy) is a common nervous system disease, the reason is that the abnormal discharge of neurons in the brain (mainly the temporal lobe) leads to transient dysfunction of the brain, which can lead to generalized or partial seizures in patients, such as sudden psychosis Activity interruption, loss of consciousness, general spasm, foaming at the mouth, urinary incontinence, bluish complexion, dilated pupils and other symptoms, the onset time ranges from a few seconds to tens of seconds. Epileptic seizures can cause direct or indirect physical damage, and bring great pain and severe mental pressure to patient...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0793C12Q1/02
Inventor 刘猛六柳明杰沈雁飞
Owner 宁波易赛腾生物科技有限公司
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