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A method for activating erythropoietin gene expression

A technology of erythropoietin and gene expression, applied in gene therapy, cells modified by introducing foreign genetic material, pharmaceutical formulations, etc., can solve problems such as erythrocytosis and pure red blood cell regeneration disorder

Active Publication Date: 2018-03-27
SHANGHAI GENECHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional EPO injections are prone to erythrocytosis, and multiple injections have adverse reactions such as pure red cell aplasia (Rossert J, Pure Red Cell Aplasia Global Scientific Advisory Board (GSAB). Erythropoietin-induced, antibody-mediated pure red cell aplasia. Eur JClin Invest. 2005; 35:95-99.)

Method used

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  • A method for activating erythropoietin gene expression
  • A method for activating erythropoietin gene expression
  • A method for activating erythropoietin gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1 can activate the construction of the recombinant plasmid of EPO gene expression

[0062] 1. Determination of the upstream target sequence of the EPO gene promoter

[0063] Find the sequence of the upstream promoter region of the EPO gene through NCBI database analysis (the sequence number of the EPO gene is: NM_000799.2, the sequence of the upstream promoter region of the EPO gene is shown in SEQ ID NO: 1), and import it into https: / / boglab.plp.iastate.edu / analysis.

[0064] The target sequence of the TAL effector of the EPO gene is: ACCCCTGGCGACCCCTCA (SEQ ID NO: 2), and the target recognition module corresponding to the target sequence is NI-HD-HD-HD-HD-NG-NN-NN-HD- NN-NI-HD-HD-HD-HD-NG-HD-NI; its amino acid sequence composition is shown in SEQ ID NO:21.

[0065] According to the composition of the target recognition module, the target recognition module of the EPO gene is grouped sequentially from the amino terminal to the carboxyl terminal in a group...

Embodiment 2

[0094] Example 2: Transfection of constructed TALE-TF-EPO plasmid into human MSC cells

[0095] The recombinant plasmid TALE-TF-EPO and the negative control plasmid MOCK-TALE-TF were extracted with a plasmid extraction kit from Qiagen, and prepared into a 100ng / μl storage solution. 24 hours before transfection, human MSC cells in the logarithmic growth phase were digested with trypsin, and the cell density was adjusted to 1.5×10 with DMEM complete medium containing 10% fetal bovine serum. 5 Cells / ml, seeded in 6-well plate, 37°C, 5% CO 2 Cultured in an incubator. When the cell density reaches 70%-80%, it can be used for transfection. The TALE-TF-EPO plasmid and the negative control MOCK-TALE-TF plasmid were respectively transfected into human MSC cells by liposome-mediated transfection method, and the specific operation process was according to Lipofectam TM 2000 reagent instructions, change the fresh medium after 6 hours of transfection, continue to culture for 72 hours, ...

Embodiment 3

[0096] Embodiment 3: the semiquantitative RT-PCR method detection of the EPO gene expression after transfection MSC cell

[0097] Total RNA was extracted from the MSC cells transfected with the negative control MOCK-TALE-TF plasmid and the MSC cells transfected with the TALE-TF-EPO plasmid, respectively, according to the Invitrogen Trizol operating instructions. According to Promega's M-MLV operating instructions, RNA was reverse-transcribed to obtain cDNA (see Table 9 for the reverse-transcription reaction system, react at 42°C for 1 hour, and then bathe in a water bath at 70°C for 10 minutes to inactivate the reverse transcriptase) to extract total cellular RNA Afterwards, the expression of the mRNA level of the EPO gene was detected by RT-PCR method.

[0098] The TP800 Real time PCR instrument (TAKARA) was used for real-time quantitative detection. The primers of EPO gene are as follows: upstream primer 5'-CGCTAGCGGATGGGGGTGCACGAATGT-3' (SEQ ID NO:24) and downstream primer...

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Abstract

The present invention relates to the field of biotechnology, in particular to a method for activating the expression of erythropoietin gene. In order to activate the expression of erythropoietin gene in target cells by using TALE technology, the following steps are included: 1) in the erythropoietin gene Find the TALE target sequence upstream of the promoter, and design a target recognition module composed of TAL nucleic acid recognition units that specifically recognizes the TALE target sequence; 2) Construction of the coding sequence of the target recognition module; 3) Ligation by enzyme digestion , connecting the coding sequence of the target recognition module prepared in step 2) with the backbone vector to obtain a recombinant plasmid; 4) transforming the recombinant plasmid into target cells and culturing. The invention uses TALE technology to activate the expression of erythropoietin gene in autologous bone marrow mesenchymal stem cells, provides a new cell source for gene therapy, and can play an important role in tissue engineering and cell therapy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for activating the expression of erythropoietin gene. Background technique [0002] TALE (Transcription activator-effectors) technology is a new molecular biology tool. Researchers have found that the amino acid sequence of the nucleic acid binding domain of a TALE protein secreted by the plant pathogen Xanthomonas has a constant correspondence with the nucleic acid sequence of its target site. Using the sequence modules of TALE, it can be assembled into a modular protein that specifically binds to any DNA sequence, so as to achieve the purpose of targeted manipulation of endogenous genes (Li T, Huang S, Jiang W Z, et al. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNAcleavage domain. Nucleic Acids Res. 2011; 39(1):359-372.). [0003] At present, there are two main applications of TALE technology: 1) gene knockout of TALEN (transcription activ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10A61K48/00A61P9/00
Inventor 朱向莹许可翁仕强曹跃琼金杨晟
Owner SHANGHAI GENECHEM
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