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Method for removing Sidt2 gene in Beta-TC-6 cell line based on CRISPR/Cas9 system

A beta-tc-6, px459-sidt2 technology, applied in the field of medical genetic engineering, can solve problems such as optimization of electrotransfection parameters, large differences in transfection parameters, and effects of cell state electrotransduction efficiency.

Inactive Publication Date: 2019-07-19
THE FIRST AFFILIATED HOSPITAL OF WANNAN MEDICAL COLLEGE YIJISHAN HOSPITAL OF WANNAN MEDICAL COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, electroporation transfection has great differences in transfection parameters for different cell lines, so it is necessary to optimize electrotransfection parameters for different cell lines. At present, more mature cells using electroporation are common cell lines or primary cells. , less electroporation was used for islet cell lines, and electrotransfection parameters were not optimized
In addition, the purity and concentration of the plasmid, the state of the cell, etc. will affect the efficiency of electroporation.

Method used

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  • Method for removing Sidt2 gene in Beta-TC-6 cell line based on CRISPR/Cas9 system
  • Method for removing Sidt2 gene in Beta-TC-6 cell line based on CRISPR/Cas9 system
  • Method for removing Sidt2 gene in Beta-TC-6 cell line based on CRISPR/Cas9 system

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Embodiment 1

[0028] Such as figure 1 , figure 2 , image 3 and Figure 4 As shown, the method of deleting the Sidt2 gene in the Beta-TC-6 cell line based on the CRISPR / Cas9 system, the specific experimental steps are as follows:

[0029] sgRNA oligo DNA sequence synthesis; use the sgRNA online design tool to design oligo DNA with a length of about 20bp for the mouse Sidt2 gene. CACC needs to be added to the 5' end of the F chain of the sgRNAssDNA sequence so that it can be complementary to the vector after BbsI digestion. It should be noted that The first base of the F chain must be G. If the first base of the selected Guide sequence is not G, a G can be added to enhance the activity of the U6 promoter, and AAAC is added to the 5' end of the R chain. In addition, a primer needs to be designed upstream and downstream of the site for subsequent detection of positive clones. The primers can amplify a DNA fragment of about 300 bp, the upstream primer is about 100 bp away from the mutation ...

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Abstract

The present invention discloses a method for removing a Sidt2 gene in a Beta-TC-6 cell line based on a CRISPR / Cas9 system. Aiming at characteristics that mouse insulinoma pancreatic beta-cells (Beta-TC-6) are difficult to transfect and CRISPR / Cas9 plasmid molecules are relatively large, an electroporation is used for delivery of Cas9 plasmids, and besides, different perforation voltage gradients are arranged to optimize electro-delivery parameters are arranged to realize high-efficient delivery of the CRISPR / Cas9 plasmids in the mouse insulinoma pancreatic beta-cells (Beta-TC-6); and at the same time, a CRISPR / Cas9 gene editing system is combined with the electroporation to overcome a problem that the CRISPR / Cas9 plasmids are difficult in transduction and provide a practical and feasible method for the CRISPR / Cas9 gene editing system in routine molecular biology laboratories, and the method has an extremely high value for promotion and application.

Description

technical field [0001] The present invention relates to the field of medical genetic engineering, in particular to a method for deleting Sidt2 gene in Beta-TC-6 cell line based on CRISPR / Cas9 system. Background technique [0002] The CRISPR / Cas9 gene editing system has become the third-generation gene editing technology after zinc finger proteins (ZFPs) and transcription activator-like effector nucleases (TALEs). Compared with traditional gene editing tools, it has many advantages. First, the CRISPR / Cas9 gene editing system has strong adaptability and operability. Cas9 from Streptococcus pyogenes and Streptococcus thermophilus has only 2 bases due to the PAM recognition sequence ( GG), a large number of targets can be found in almost all genes. Cas9 protein is active in almost all organisms and cells tested so far, including bacteria, yeast, plants, fish, and mammalian cells; secondly, the CRISPR / Cas9 gene editing system is easy to learn, and there are currently many mature...

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Application Information

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IPC IPC(8): C12N15/90C12N15/85C12N9/22C12N15/66
CPCC12N15/907C12N15/85C12N9/22C12N15/66
Inventor 高家林王李卓吕康甲章尧粱飞腾张杨
Owner THE FIRST AFFILIATED HOSPITAL OF WANNAN MEDICAL COLLEGE YIJISHAN HOSPITAL OF WANNAN MEDICAL COLLEGE