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Method for adjusting and controlling expression of nestin gene in neural stem cell and method of targeting cell

A neural stem cell and gene expression technology, applied in the field of gene expression regulation, can solve the problems of low efficiency, difficulty, long working time, etc., and achieve the effect of low off-target rate

Active Publication Date: 2019-07-30
珠海乐维再生医学科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, in transient expression, the exogenous DNA will not be integrated into the genome, but the transient expression can only be maintained for a few days; in stable expression, the exogenous gene is integrated with the genome and can be continuously expressed in the cell
The traditional way of establishing stable cell lines is usually through transient transfection and screening of resistance genes on gene carriers to finally obtain stable cell lines. This method has the problems of long working time and low efficiency. In addition, due to the relatively long Nestin gene sequence , it is difficult to establish a stable overexpression cell line using the traditional method, so it is necessary to establish a new gRNA target sequence for Nestin gene overexpression or silencing

Method used

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  • Method for adjusting and controlling expression of nestin gene in neural stem cell and method of targeting cell
  • Method for adjusting and controlling expression of nestin gene in neural stem cell and method of targeting cell
  • Method for adjusting and controlling expression of nestin gene in neural stem cell and method of targeting cell

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Experimental program
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Effect test

Embodiment 1

[0031] A method for regulating nestin gene expression in neural stem cells specifically includes the following steps in sequence:

[0032] First design the gRNA of the nestin gene.

[0033] The cDNA sequence of the nestin gene is shown in the reference sequence 30, and the amino acid sequence of the NESTIN protein is shown in the reference sequence 31. For the sequence of the nestin gene, 12 pairs of gRNAs are designed near the transcription start of the nestin gene, including 6 pairs of guide transcription repressors. Nestin-i gRNA and 6 pairs of nestin-a gRNA guiding transcription activators, wherein the sequence of nestin-i gRNA is shown in sequence 1-12, and the sequence of nestin-a gRNA is shown in sequence 13-24. Nestin-i gRNA is used to guide the transcription repressor to bind to the target site downstream of the transcription start site of the first exon of the nestin gene. nestin-agRNA is used to guide the transcription activator to bind to the target site upstream of th...

Embodiment 2

[0107] Detection of nestin gene expression level.

[0108] 1. Extracting total RNA from cell lines, specifically including the following steps:

[0109] 1) Aspirate and discard the culture medium in the culture dish and add 2ml PBS to rinse the cells. After discarding the PBS, add 1ml TRIZOL solution, and incubate on ice for 10 minutes;

[0110] 2) Transfer the solution with TRIZOL added in step (1) to a centrifuge tube, add 200 μl of chloroform and vortex to mix evenly, and incubate on ice for 5 minutes;

[0111] 3) Centrifuge the centrifuge tube at 4°C and 13000g for 15 minutes. After centrifugation, carefully transfer the upper aqueous phase to another centrifuge tube;

[0112] 4) Add an equal volume of isopropanol to the centrifuge tube, and after mixing, put the centrifuge tube at -80°C for precipitation for 30 minutes;

[0113] 5) Centrifuge the centrifuge tube at 13000g at 4°C for 15 minutes. A white precipitate can be seen at the bottom of the centrifuge tube. At this time, the s...

Embodiment 3

[0139] Detection of NESTIN protein level

[0140] First extract the total protein of the cells, and then perform Western Blot. Specifically include the following steps:

[0141] 1) After rinsing the cell line collected in Example 1 with PBS, add protein lysis solution and lyse on ice for 15 minutes;

[0142] 2) Use an ultrasonic instrument to ultrasonically disrupt the mixture in step (1) to fully lyse the cells;

[0143] 3) Centrifuge the centrifuge tube at 4°C and 13000g for 15 minutes, and transfer the supernatant protein to a new centrifuge tube;

[0144] 4) Use the Bradford protein concentration determination kit produced by Solarbio to determine the protein concentration. The product model of the Bradford protein concentration determination kit is PC0010;

[0145] 5) Take 50μg of total protein and add it to 20μl 2xSDS loading buffer, and add ddH 2 O make the total volume 40μl, heat it in boiling water for 10min;

[0146] 6) Take 35μl of the denatured sample in step (5) for SDS-PAGE...

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Abstract

The invention discloses a method for adjusting and controlling expression of a nestin gene in a neural stem cell and a method of targeting a cell, and relates to the technical field of adjustment andcontrol over gene expression. The method for adjusting and controlling the expression of the nestin gene in the neural stem cell comprises the following successively-conducted steps that firstly, gRNAof the nestin gene is designed nearby transcription initiation of the nestin gene, then a carrier is constructed, and the gRNA of the nestin gene can be any one of nestin-i gRNA for guiding a transcriptional repressor and nestin-a gRNA for guiding a transcriptional activator. The method for adjusting and controlling the expression of the nestin gene in the neural stem cell is simple, low in off-target rate and capable of achieving stable expression in cell strains. The defects generated from construction of stable cell strains by a traditional method are overcome, and the stable cell strainscan be obtained in a short time.

Description

Technical field [0001] The present invention relates to the technical field of gene expression regulation, in particular to a method for regulating the expression of nestin gene in neural stem cells and a method for targeting cells. Background technique [0002] The protein coding product of nestin gene is NESTIN. NESTIN is a type 6 intermediate filament protein, also known as nestin. Its molecular weight is 220KDa-250KDa, and it is located on chromosome 1. The nestin gene is composed of 3 introns and 4 exons in structure. NESTIN protein is mainly divided into N-terminal (1-7aa), α-helical region (8-313aa) and C-terminal (314-1621aa) in structure. The α-helical region is subdivided into 4 parts, 1A and 1B. , 2A and 2B. [0003] Nestin gene is widely expressed during embryonic development, and its earliest expression is in the single-cell neuroectoderm of the embryo. In addition, nestin is also expressed in a variety of cells, such as neovascular endothelial cells, mesorenal mes...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/90C12N5/10
CPCC07K14/47C12N15/113C12N15/907C12N2310/10C12N2310/20
Inventor 黄国滨周红艳李爱群鲁浩龚春丽郑妍明周珊珊
Owner 珠海乐维再生医学科技有限公司
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