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Synthetic modified Olig2 mRNA and application thereof

A technology of sequencing and oligodendrocytes, applied in the direction of genetically modified cells, medical preparations containing active ingredients, applications, etc., can solve problems such as limiting clinical application, achieve high induction differentiation ability, rapid and efficient directed differentiation Effect

Pending Publication Date: 2021-08-20
SUN YAT SEN UNIV SHENZHEN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method also has obvious disadvantages
For example, exogenous SOX10 is integrated into the genome of neural precursor cells based on lentiviral vectors, so that the induced oligodendrocytes may have the risk of gene integration and insertional mutagenesis, which greatly limits its clinical application. use

Method used

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  • Synthetic modified Olig2 mRNA and application thereof
  • Synthetic modified Olig2 mRNA and application thereof
  • Synthetic modified Olig2 mRNA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] A synthetically modified Olig2 mRNA using a strategy such as figure 1 Shown, prepared by the following method:

[0068] 1. Gene editing.

[0069] By site-directed mutagenesis, the 147th serine codon UCC of the Olig2 protein coding region was mutated into alanine codon GCC, GCG, GCA or GCU by conventional methods.

[0070] 2. In vitro transcription.

[0071] The above mutated DNA was synthesized into Olig2 by in vitro transcription WT mRNA and Olig2 S147A mRNA, the specific operation is as follows:

[0072] Using the LR reaction protocol kit (Thermo Fisher, USA), the gene open reading frames of wild-type Olig2 and mutated Olig2 were cloned into the vector PCR2-UTR-R1R2, treated with restriction endonucleases, and linearized, Obtain the open reading frame of the target gene. Using the linearized vector as a template, carry out tail-PCR reaction with the 5' end T7 polymerase promoter primer and the 3' end UTR long T tail primer. The PCR products were used as templ...

Embodiment 2

[0074] A method for promoting the differentiation of human induced pluripotent stem cells to oligodendrocytes, comprising the following steps:

[0075] 1. Single cell subculture of human induced pluripotent stem cells

[0076] 1.1 Use D-PBS containing calcium and magnesium ions to dilute the COAT-1 coating solution at a ratio of 1 / 20, coat 6-well plate with 1ml per well, and incubate in a constant temperature incubator at 37°C for at least half an hour after coating;

[0077] 1.2 Human induced pluripotent stem cells were cultured in Cellartis DEF-CS single cell medium, digested with TrypLEExpress Enzyme, and incubated in a constant temperature incubator at 37°C for 5 minutes;

[0078]1.3 After the digestion is completed, use the medium to stop the digestion, centrifuge and wash, resuspend in Cellartis DEF-CS medium, and follow the 3×10 5 The number of cells per plate was seeded in pre-coated six-well plates for further culture.

[0079] 1.4 Human induced pluripotent stem cel...

Embodiment 3

[0100] The marker genes and corresponding surface protein markers of induced pluripotent stem cell-derived oligodendrocytes and precursor cells were identified by fluorescent quantitative PCR and cellular immunofluorescence.

[0101] 1. Fluorescent quantitative PCR identification

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Abstract

The invention relates to synthetic modified Olig2mRNA and application thereof, and belongs to the technical field of stem cell induced differentiation. The protein translated by the Olig2mRNA is always in a dephosphorylation state. By transfecting neural precursor cells derived from human induced pluripotent stem cells, rapid and efficient directional differentiation of the human induced pluripotent stem cells to oligodendrocytes is realized, and the designed and modified Olig2mRNA shows higher efficiency of induced differentiation than wild type Olig2mRNA. By utilizing the method disclosed by the invention, the induced pluripotent stem cells can be quickly differentiated into the oligodendrocytes, the risk of gene integration of the oligodendrocytes induced based on overexpression of a transcription factor SOX10 in the past can be avoided, and the method is more beneficial to the development of clinical tests. The rapid induction of oligodendrocytes derived from the human induced pluripotent stem cells provides a technical support for treating myelin sheath injury type neurodegenerative diseases through transplantation of oligodendrocyte precursor cells in the future.

Description

technical field [0001] The invention relates to the technical field of stem cell induction and differentiation, in particular to a synthetically modified Olig2 mRNA and its application. Background technique [0002] Oligodendrocytes are an important type of glial cells in the human brain. They wrap around the axons of neurons by forming a dense myelin sheath structure, and play a role in neuronal signal transduction, nutritional support, and maintenance of the brain. plays an important role in homeostasis. As early as the 20th century, the Brazilian scientist Hortega first proposed oligodendrocytes, but until now, the understanding of the origin and function of oligodendrocytes and their progenitors is still insufficient. [0003] For a long time, the main function of oligodendrocytes was limited to the formation and maintenance of myelin. However, more and more evidences show that oligodendrocytes have diverse effects on the central nervous system. One of the more signifi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/10C12N15/88C12N5/10A61K35/30A61P25/28
CPCC07K14/4702C12N15/102C12N15/88C12N5/0622C12N5/0696A61K35/30A61P25/28C12N2510/00C12N2506/45C12N2501/60
Inventor 邓文斌徐健聂怡初麦扬张祎迪赵景新蒋近军萧倩谢芫
Owner SUN YAT SEN UNIV SHENZHEN
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