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Nrf1β gene directional knockout recognition sequence pair, talens, vector pair and application in human hepatocytes

A hepatocyte and beta gene technology, applied in vectors, nucleic acid vectors, genetic engineering, etc., can solve the problems of off-target siRNA technology, poor stability and repeatability of siRNA technology, and cumbersome operation, and achieve simple, easy, high-quality gene editing. The effect of gene editing, the effect of simple and easy research

Active Publication Date: 2020-05-22
CHONGQING UNIV
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Problems solved by technology

[0003] In traditional research methods, the principle of using siRNA interference technology to study the function of Nrf1 is to reduce the level of Nrf1 protein in cells by degrading the mRNA of Nrf1 in cells, and Nrf1 in cells cannot be completely eliminated, so the research results obtained cannot exclude The impact of residual Nrf1 protein, in addition, siRNA technology also has off-target problems, and the stability and reproducibility of siRNA technology are poor; the use of homologous recombination system gene knockout technology to study Nrf1 is to knock out all Nrf1 genes in the genome, However, this method is cumbersome to operate, and the longer knockout fragments on the genome may lead to uncontrolled transcription, and cannot distinguish the functions of different Nrf1 subunits

Method used

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  • Nrf1β gene directional knockout recognition sequence pair, talens, vector pair and application in human hepatocytes
  • Nrf1β gene directional knockout recognition sequence pair, talens, vector pair and application in human hepatocytes
  • Nrf1β gene directional knockout recognition sequence pair, talens, vector pair and application in human hepatocytes

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Effect test

Embodiment 1

[0034] Example 1 Selection of editing site and design and construction of Nrf1β-TALEN plasmid

[0035] According to the gene editing principle of TALEN technology, the Nrf1β translation start codon in the CDS region of the Nrf1 genome was selected as the Nrf1β gene TALEN target editing site, and the sequence was: 5'-tagtgaacagtggtgctcttctcccctcccaggcc gaagtgaacacatcagcaagtgaaatcctgtacag-3' (bold font is the Nrf1β protein translation initiation codon). Among them, the left arm of TALEN Nrf1β-Talen L recognizes 17 nucleic acid base sequences as 5'-tggtgctcttctcccct-3' (SEQ ID NO.1); the right arm Nrf1β-Talen R recognizes 18 nucleic acid base sequences as 5'-tttcacttgctgatgtgt- 3' (SEQ ID NO.2); the 18bp sequence between the two recognition and binding sites is 5'-cccaggccatggaagtga-3', which is the cleavage site for the non-specific endonuclease Fok1 dimer. ( figure 1 )

[0036] According to the nucleic acid bases recognized by the left and right arms of the Talen, according...

Embodiment 2

[0042] Screening, cultivation and identification of monoclonal cell lines knocked out of the Nrf1β gene in embodiment 2

[0043] 1) Transfection of HL7702 cells with Nrf1β-TALEN plasmid:

[0044] a. HL7702 cells were planted in 2 wells of a six-well plate, respectively recorded as A (plasmid transfection group) and B well (control group), with 300,000 cells per well, and cultured overnight in DMEM medium. After the cells grow at the bottom of the well plate until about 80% of the bottom plate is covered, prepare to transfect the Nrf1β-TALEN plasmid.

[0045] b. Aspirate the DMEM medium in wells A and B, and add 800 μL Opti-MEM.

[0046] c. Prepare solution 1, solution 2 and solution 3; among them, the formula of solution 1 is as follows: take 1.5 μg of Nrf1β-TALEN L plasmid and 3 μg of Nrf1β-TALEN R plasmid, add 100 μL Opti-MEM, mix well and let stand for 5 minutes;

[0047] Solution 2: 100 μL Opti-MEM;

[0048] Solution 3: Take 18 μL of lipo20000 and add 200 μL Opti-MEM, m...

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Abstract

The invention provides an identification sequence pair for targeted knockout of Nrf1beta gene in human hepatocyte, Talens, a carrier pair and an application. The identification sequence pair comprises a left arm identification sequence and a right arm identification sequence, wherein a nucleotide sequence of the left arm identification sequence is as shown in SEQ ID NO.1; a nucleotide sequence of the right arm identification sequence is as shown in SEQ ID NO.2; the nuclease Talens comprises a nuclease left arm Nrf1beta-Talen L for identifying the nucleotide sequence of SEQ ID NO.1 and a nuclease right arm Nrf1beta-Talen R for identifying the nucleotide sequence of SEQ ID NO.2; the carrier pair comprises an Nrf1beta-Talen L carrier and an Nrf1beta-Talen R carrier; and the carrier pair can be applied to directed knockout of the Nrf1beta gene in a human hepatocyte line and construction of an Nrf1beta gene knockout type human hepatocyte line.

Description

technical field [0001] The invention belongs to the technical field of gene knockout, and specifically relates to a recognition sequence pair, a Talens, a vector pair and an application thereof for the directional knockout of Nrf1β gene in human liver cells. Background technique [0002] Nuclear factor E2-related factor 1 (TCF11 / Nrf1) has many important physiological functions such as anti-oxidation, anti-inflammation, regulation of protein degradation, and balance of glucose and lipid metabolism. However, due to the existence of multiple subtypes of Nrf1 protein in cells, including Nrf1α, Nrf1β, Nrf1χ, Nrf1D, etc., and various post-translational modifications, the function of Nrf1 has not been thoroughly studied. Hepatic cell lines are good materials for studying the regulation of glucose and lipid metabolism by Nrf1. At present, there is no cell line knocked out of human liver cell line Nrf1 for sale, which makes the specific molecular mechanism and signaling pathway of Nr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/85C12N5/10
CPCC07K14/4702C12N9/22C12N15/11C12N15/85C12N2310/10C12N2800/107C12N2800/80
Inventor 张义国邱露
Owner CHONGQING UNIV
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