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Method for preparing CRX mutation related retinopathy non-human mammal model

A technology for non-human mammals and retinopathy, applied in the fields of molecular biology and biomedicine, which can solve the problems of technical difficulty, high cost of animal models, inability to apply mutation sites, etc., and achieve the effect of simple construction

Inactive Publication Date: 2020-11-24
上海朗昇生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the establishment of animal models carrying specific pathogenic mutations is costly and technically difficult, and cannot be applied to all mutations

Method used

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  • Method for preparing CRX mutation related retinopathy non-human mammal model
  • Method for preparing CRX mutation related retinopathy non-human mammal model
  • Method for preparing CRX mutation related retinopathy non-human mammal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] This example provides a method for establishing a CRX mutation-related retinopathy model in wild-type mice using recombinant AAV.

[0055] 1. Materials

[0056] AAV-GFP vector, plasmid pAAV-RC, pHelper; HEK293T cells, Y79 human retinoblastoma cells; C57BL / 6 mice.

[0057] 2. Method

[0058] (1) Construction of adeno-associated virus vector

[0059] The c.611delC (p.S204fs) mutant sequence of the CRX gene was cloned in vitro, and the AAV-GFP vector was used as the starting plasmid. After a series of conventional enzyme digestion and ligation, the CRX mutant gene carrying the HA tag was used to replace the GFP sequence, and the expression of the CRX mutant gene was constructed. Adeno-associated virus vector AAV-CRX-mut-HA. Similarly, the wild-type CRX virus vector AAV-CRX-wt-HA carrying the HA tag was constructed.

[0060] The gene sequence is as follows:

[0061] CRX-mut-HA sequence:

[0062] ATGATGGCGTATATGAACCCGGGGCCCCACTATTCTGTCAACGCCTTGGCCCTAAGTGGCCCCAGTGTGGATC...

Embodiment 2

[0076]Application of recombinant AAV to establish a model of CRX mutation-associated retinopathy in wild-type mice.

[0077] 1. Materials

[0078] Mouse model established in Example 1: mice injected subretinal with AAV-CRX-wt-HA and AAV-CRX-mut-HA virus vectors respectively.

[0079] 2. Method

[0080] In situ detection of apoptosis in the outer nuclear layer by TUNEL staining

[0081] After 4 weeks of modeling, mouse eyeballs were taken for frozen sections. According to the instructions of the Roche in situ cell death detection kit, TUNEL staining was performed, and the number of apoptotic cells in multiple fields of view was recorded and counted.

[0082] Detection of mRNA levels of CRX downstream target genes in mouse retina by QPCR

[0083] The RNA extraction kit was used to extract the total RNA in the mouse retinal homogenate, and then the first strand of cDNA was synthesized by reverse transcription using a reverse transcription kit. A real-time fluorescence quanti...

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Abstract

The invention relates to the field of molecular biology and biomedicine, in particular to a method for preparing a CRX mutation related retinopathy non-human mammal model. The method comprises the step of expressing CRX[S204fs] protein in retina photoreceptor cells of an animal by adopting a transgenic method. The CRX[S204fs] protein has a competitive antagonism effect (type-III mutation) on wildtype protein, so that retinopathy symptoms can be shown as long as the CRX[S204fs] protein is expressed in the animal body, a wild type CRX gene does not need to be knocked out, and construction of the animal model is simpler, quicker and more economical.

Description

technical field [0001] The invention relates to the fields of molecular biology and biomedicine, in particular to a method for preparing a non-human mammalian model of CRX mutation-related retinopathy. Background technique [0002] The cone-rod homeobox gene (CRX) encodes photoreceptor cell-specific transcription factors, which can transcribe and activate various photoreceptor cell-specific genes such as rhodopsin and retinol binding protein. It plays a crucial role in the differentiation, development and integrity of rod cells. CRX is a common pathogenic gene in hereditary retinopathy, and its mutation can cause a variety of diseases, including Leber's congenital amaurosis, cone-rod dystrophy (CoRD), and cone-rod dystrophy , retinitis pigmentosa and macular dystrophy. Approximately one-third of CRX pathogenic variants cause CORD, which is characterized by progressive cone dysfunction manifested primarily by loss of central vision, abnormal color vision, and optic nerve dy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864A01K67/027A61K49/00
CPCA01K67/0278A01K2217/072A01K2227/105A01K2267/03A61K49/0008C07K14/47C12N15/86C12N2750/14143
Inventor 汪枫桦
Owner 上海朗昇生物科技有限公司
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