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Tyrosinemia type I monkey model and establishment method and application thereof

A technology of tyrosinemia and establishment method, which is applied in the field of tyrosinemia type I monkey model and its establishment, and can solve the problems that the FAH gene knockout cynomolgus monkey model has not been reported.

Active Publication Date: 2019-09-20
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the FAH gene knockout cynomolgus monkey model has not been reported

Method used

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  • Tyrosinemia type I monkey model and establishment method and application thereof
  • Tyrosinemia type I monkey model and establishment method and application thereof
  • Tyrosinemia type I monkey model and establishment method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] A type I monkey model of tyrosinemia is established by the following method:

[0068] 1. Analyze and design the FAH gene, use the exon EXON1 and / or EXON2 as the cutting site, construct the TALEN targeting modification gene vector plasmid, and synthesize the TALEN mRNA; the details are as follows:

[0069] (1) Target selection.

[0070] Through the analysis of the FAH gene sequence, 3 pairs of TALEN targeting vectors were designed. The base sequence of the target recognition is 16-17bp, and the spacer sequence is between 14-16bp; the targeting cutting sites are selected at the front On the exons EXON1 and EXON2, in order to form a frameshift or mutation of the DNA coding sequence to achieve the purpose of gene knockout.

[0071] The designed TALEN left and right arm sequences targeting the Fah gene:

[0072] TALEN-T1 left arm: GCTCGCCAGCATGTCCT (SEQ ID NO.1);

[0073] TALEN-T1 right arm: GGGGAAGTCGGAATCCT (SEQ ID NO.2);

[0074] TALEN-T2 left arm: CCACAACCTGCCCTAC (S...

Embodiment 2

[0135] Detection of liver histopathology and physiological indicators in FAH gene knockout cynomolgus monkeys.

[0136] (1) Immunohistochemistry:

[0137] a) Paraffin sections were placed in an oven at 60°C for 2 hours, and immediately placed in xylene for elution three times, 10 minutes each time; followed by gradient alcohol: 100%, 5 minutes; 95%, 5 minutes; 80%, 5 minutes; 70%, 5 minutes ; Put it into distilled water twice, 10min / time.

[0138] b) Antigen retrieval: add citrate buffer, heat to 96° C. for 15 minutes, and cool at room temperature.

[0139] c) Add hydrogen peroxide to block endogenous peroxidase, keep away from light at room temperature for 10 minutes, wash with PBS twice, 5 minutes each time;

[0140] d) Add goat serum dropwise to block for 10 minutes; add appropriate concentration of primary antibody and incubate overnight;

[0141] e) Wash with PBS twice, 5 min each time; according to the operation instructions of Maixin immunohistochemistry and DAB colo...

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Abstract

The invention relates to a tyrosinemia type I monkey model and an establishment method and an application thereof, and belongs to the technical field of medical animal disease model. The method includes the steps of design of a TALEN target of an FAH gene, construction of an FAH gene-knockout TALEN plasmid, acquisition and screening of gene targeting RNA fragments and preparation of the FAH gene-knockout monkey model. The FAH gene-knockout monkey model is obtained finally, and the genotype identification analysis and physiological and pathological phenotype analysis on the model are carried out, so the disease performance of patients with human tyrosinemia type I are clarified and the success of the model construction is confirmed. Establishment of the model can provide a reliable standard animal model for the follow-up development of study and clinical transformation of gene therapy and cell therapy of hereditary metabolic deficiency diseases, and also provides an animal model more close to the patients for screening of traditional drugs and evaluation of efficacy and safety.

Description

technical field [0001] The invention relates to the technical field of medical animal disease models, in particular to a type I monkey model of tyrosinemia and its establishment method and application. Background technique [0002] Tyrosinemia type I (hereditary tyrosinemia type 1, HT1) (OMIM276700) is a typical inherited metabolic defect (Inborn errors of metabolism, IEM) disease. HT1 is caused by a mutation of the fumarylacetoacetate hydrolase (FAH) gene, which is an autosomal recessive genetic disease. The sequence encoding the gene is located at 15q23-q25 and contains 14 exons. A total of 96 exons have been discovered so far. FAH gene mutations, nearly 84 of which are pathogenic genotypes. This gene is the last metabolic enzyme of the tyrosine metabolic pathway, and its deletion leads to a large accumulation of tyrosine and succinylacetone in the body, causing a hypertyrosinemia phenotype, causing serious damage to the body and threatening life. Increased blood or urin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N15/55A01K67/027A61K49/00
CPCC12N15/907C12N9/14C12Y307/01002A01K67/0276A61K49/0008A01K2217/075A01K2227/106A01K2267/0306
Inventor 项鹏柯琼赖兴强陈洪李伟强毛富祥
Owner SUN YAT SEN UNIV
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