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Promoter specifically expressed in retinal Muller cell and application thereof

A promoter, retinal technology, applied in the field of biomedicine, can solve the problems of low drug utilization, adverse reactions, retinal folds, etc.

Pending Publication Date: 2022-04-15
SICHUAN ACADEMY OF MEDICAL SCI SICHUAN PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of vitreous and retinal injections of drugs has the problem of low drug utilization. Repeated injections will lead to complications such as retinal folds, or adverse reactions due to drug side effects

Method used

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  • Promoter specifically expressed in retinal Muller cell and application thereof
  • Promoter specifically expressed in retinal Muller cell and application thereof
  • Promoter specifically expressed in retinal Muller cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1, the synthesis of Müller-P promoter of the present invention

[0024] The DNA oligonucleotide sequence (SEQ ID NO.1) was synthesized according to the DNA sequence template using DNAchem technology.

Embodiment 2

[0025] Embodiment 2, the preparation of recombinant adeno-associated virus of the present invention

[0026] The target gene (Example 1) was cloned into the adeno-associated virus vector to obtain the recombinant virus, which was transfected into HEK293T cells for virus packaging. After 72 hours of transfection, the cells produced a large amount of recombinant virus, and then the culture supernatant and cell pellet were harvested respectively. PEG8000 precipitates the virus in the medium supernatant, lyses the cell pellet, and pools the virus obtained from the cell pellet and supernatant. Finally, the virus is purified with iodixanol to obtain the purified recombinant adeno-associated virus carrying the Müller-P promoter of the present invention.

Embodiment 3

[0027] Example 3, Preparation of recombinant adeno-associated virus of the present invention

[0028] The target gene (Example 1) and the fluorescent protein GFP gene were cloned into the adeno-associated virus vector to obtain the recombinant virus, which was transfected into HEK293T cells for virus packaging. After 72 hours of transfection, the cells produced a large amount of recombinant virus, and then harvested respectively on the culture medium. Supernatant and cell pellet, using PEG8000 to precipitate the virus in the medium supernatant, lysing the cell pellet, and merging the virus obtained from the cell pellet and supernatant. Finally, the virus is purified with iodixanol to obtain the purified recombinant adeno-associated virus carrying the Müller-P promoter of the present invention.

[0029] The beneficial effects of the present invention are demonstrated through experimental examples below.

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Abstract

The invention provides a promoter Muller-P which is specifically expressed in a retinal Muller cell, and the sequence of the promoter Muller-P is as shown in SEQ ID NO. 1. The promoter disclosed by the invention has very strong activity of specific expression in a retinal Muller cell, and the recombinant adeno-associated virus based on the promoter has potential clinical application value in gene therapy of retinal diseases related to the Muller cell. Besides, the promoter provided by the invention can specifically express a green fluorescent protein GFP gene in the Muller cell, can realize positioning of the Muller cell and fluorescence labeling of the Muller cell, can be used for screening the Muller cell, and has potential application value in researches such as Muller cell transdifferentiation.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a promoter specifically expressed in retinal Müller cells and its application. Background technique [0002] The retina is a part of the central nervous system. The mammalian central nervous system includes two types of cells: neurons and glial cells. Among them, Müller cells are a kind of glial cells unique to the retina. It was created by the German anatomist Heinrich Mu Le (Heinrich Müller) in 1851 discovered in the retina, and named after the discoverer. Retinal Müller cells span the entire retina, extending from the inner limiting membrane to the outer nuclear layer, forming a dense and regular pattern, accounting for about 90% of all retinal cells, and playing an important role in maintaining the shape and physiological functions of the retina. Studies have found that the morphology, physiological function and metabolism of Müller cells will undergo many changes in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/864C12N5/10C12Q1/02C12N15/12
Inventor 黄璐琳杜明燕
Owner SICHUAN ACADEMY OF MEDICAL SCI SICHUAN PROVINCIAL PEOPLES HOSPITAL
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