Construction method and application of ARHGAP11B gene deleted iPSC cell line

A construction method and cell line technology, applied in microorganism-based methods, genetically modified cells, artificially induced pluripotent cells, etc., can solve the problems of constructing and knocking out induced pluripotent stem cells ARHGAP11B gene, etc., and achieve gene improvement. Editing Efficiency, Efficiency Improvement, and Complete Knockout Effect

Pending Publication Date: 2022-05-10
WUYI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There is no report on the construction of ARHGAP11B gene knockout in induced pluripotent stem cells

Method used

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  • Construction method and application of ARHGAP11B gene deleted iPSC cell line
  • Construction method and application of ARHGAP11B gene deleted iPSC cell line
  • Construction method and application of ARHGAP11B gene deleted iPSC cell line

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Example 1. A construction method for knocking out the ARHGAP11B gene in iPSC cell lines based on the CRISPR / Cas9 system

[0048] 1. sgRNA design

[0049] "sgRNA", that is, Single guide RNA, single guide RNA, can recognize specific genome fragments during CRISPR / Cas9 gene editing (including gene knockout). According to the principle of sgRNA target sequence design, the target sequence was designed in exon 6 of the human ARHGAP11B gene (the nucleotide sequence is shown in SEQ ID No.1), and three sgRNAs were constructed, namely sgRNA1, sgRNA2 and sgRNA3, with nucleotide sequences As shown in SEQ ID No.2-4 respectively (see Table 1), the sequence diagrams of the three sgRNAs are shown in figure 1 shown.

[0050] Table 1

[0051] sgRNA ID Nucleotide sequence sgRNA1 gcgtgtaccagactttatcc sgRNA2 ggaaaagataccagccatgt sgRNA3 gactttatcctggaaaagat

[0052] 2. Construction of CRISPR / Cas9 vector

[0053] The three sgRNAs were annealed to form...

Embodiment 2

[0142] Example 2. Identification and analysis of pluripotency of iPSC cell lines with ARHGAP11B gene deletion

[0143] (1) Cell immunofluorescence

[0144] The cells (iPSC63, wild-type WT iPSC) were respectively passaged to three wells of a four-well plate, and the immunofluorescence experiment was started after the cell density reached 70%-80%.

[0145] 1) Aspirate the culture medium in the four-well plate, soak it with PBS for 3 times, each time for 10 min;

[0146] 2) Fix the cells with 4% paraformaldehyde for 20 minutes, soak in PBS for 3 times, 10 minutes each time;

[0147] 3) Add 0.5% Trition X-100 (prepared in PBS) to permeate at room temperature for 20 minutes, soak in PBS for 3 times, each time for 10 minutes;

[0148] 4) Drain the PBS, add dropwise normal goat serum, and block at room temperature for 30 minutes;

[0149] 5) Blot up the blocking solution, directly add 200 μl of diluted primary antibody (Oct4, Sox2, Nanog) to each well, and incubate overnight at 4°...

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Abstract

The invention relates to the field of cell gene engineering, and particularly discloses a construction method and application of an ARHGAP11B gene deleted iPSC (induced pluripotent stem cell) cell line. According to the present invention, the targeting site of the specific sgRNA for targeted knockout of the human ARHGAP11B gene on the human ARHGAP11B gene is located on the No.6 exon of the human ARHGAP11B gene; the specific sgRNA provided by the invention can efficiently target the human ARHGAP11B gene, and a method for knocking out the ARHGAP11B gene in an iPSC cell line is constructed based on a CRISPR / Cas9 system, and the method is simple and convenient to operate and more thorough in knockout effect.

Description

technical field [0001] The invention relates to the field of cell genetic engineering, in particular to a construction method and application of an ARHGAP11B gene-deleted iPSC cell line. Background technique [0002] Mammals have a highly developed cerebral cortex, and the newly developed cerebral cortex plays an important role in regulating functions. Although the subcortical brain and spinal cord have also developed, they are functionally subordinate to the cerebral cortex . The cerebral cortex is the highest-level center that regulates or controls body movements. The human cerebral cortex has made a new leap, with the ability of abstract thinking, and has become the material basis of conscious activities. The cerebral cortex plays a vital role in memory, attention, thinking, language, consciousness, perception and more. So it can be said that the cerebral cortex is equivalent to the "headquarters" of the human body. Therefore, the research on factors affecting the deve...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N5/10C12N15/65C12N15/12C12R1/91
CPCC12N15/113C12N15/85C12N15/65C12N5/0696C07K14/4706C12N2310/20C12N2510/00C12N2800/107
Inventor 周小青刘玉陈涛金银戈郑伟邹庆剑唐成程陈敏程令印郑雨龄郑淑文郭素琴徐巨才陈静赖良学
Owner WUYI UNIV
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