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Method for activating expression of erythrogenin genes

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

Active Publication Date: 2014-03-26
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 J Clin Invest. 2005;35:95-99.)

Method used

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  • Method for activating expression of erythrogenin genes
  • Method for activating expression of erythrogenin genes
  • Method for activating expression of erythrogenin genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1 The construction of the recombinant plasmid capable of activating 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, and 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-ND-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 starts from the amino terminal to the carboxyl terminal, and is sequentia...

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 RNA from cells Afterwards, the expression of the mRNA level of the EPO gene was detected by RT-PCR method.

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

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Abstract

The invention relates to the field of biotechnology, and particularly relates to a method for activating the expression of erythrogenin genes, namely, activating the expression of erythrogenin genes in target cells by using a TALE technology. The method comprises the following steps: 1) searching a TALE target spot sequence at the upstream of an erythrogenin gene promoter, and designing a target spot identification module which is used for specifically identifying the TALE target spot sequence and composed of TAL nucleic acid identification units; 2) building a coding sequence of the target spot identification module; 3) connecting the coding sequence of the target spot identification module prepared in the step 2) with a skeleton carrier by using an enzyme digestion and connection method so as to obtain a recombinant plasmid; 4) transforming the recombinant plasmid to a target cell and culturing the cell. According to the invention, the expression of erythrogenin genes is activated in autologous mesenchymal stem cells by using a TAL technology, so that the method provides a new source of cells 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 activa...

Claims

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Application Information

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Patent Type & Authority Applications(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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