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Construction method, composition, kit and application of disease models related to abnormal glyoxylic acid metabolism

A technology of abnormal metabolism and construction method, which is applied in the field of construction of disease models related to abnormal glyoxylate metabolism, can solve problems such as inability to simulate, aggravate negative effects, and inability to simulate etiological characteristics, and achieve the effect of preventing and delaying the formation of stones

Active Publication Date: 2021-09-24
XIN HUA HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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Problems solved by technology

This stone animal model induced by compounds alone cannot simulate the etiological characteristics of the AGXT gene mutation in clinical PH1 patients, and cannot simulate the hyperoxaluria caused by abnormal liver metabolism and intermediate metabolic pathways caused by PH1 mutations in the AGXT gene and urinary system stones and other pathological manifestations; on the other hand, the use of excessive lithotropic agents will cause unpredictable toxic effects on other organs, and long induction time and high induction dose will inevitably aggravate the above negative effects

Method used

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  • Construction method, composition, kit and application of disease models related to abnormal glyoxylic acid metabolism
  • Construction method, composition, kit and application of disease models related to abnormal glyoxylic acid metabolism
  • Construction method, composition, kit and application of disease models related to abnormal glyoxylic acid metabolism

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Embodiment 1

[0063] The corresponding SGRNA is designed with the CRISPR / Cas9 gene editing system, and the corresponding SGRNA is designed for the 4th exon (near 205th amino acid) of the rat AgXT gene, and the SSODN is a template, and the homologous recombination is used to carry P. The humanized sequence of the D205N site mutation replaces the original sequence of rats and does not change the amino acid sequence. See the design strategy for constructing pH1 rats with CRISPR / CAS9 figure 2 .

[0064] This embodiment constructs an AGXT mutation rat product system through the CRISPR / CAS9 system, and the operation is as follows:

[0065] (1) We first use the CAS9 system and a precisely designed homologous sequence to introduce a specific point mutation in the rat AgXT gene, corresponding to human first 183 amino acid residual aspartate (205 in rat AgXT) Bit) and the other three mutations nearby (cause the genomic sequence of the region to be consistent with human, do not change the encoded am...

experiment example 1

[0078] Agxt D205N Characterization of rat strains PH1:

[0079] Gene functions (1) Agxt characterization:

[0080] First, we tested the homozygous Agxt obtained in Example 1 D205N Agxt gene expression in rats. Since D205N mutations affecting mRNA stability, using real-time quantitative PCR (primer sequences are shown in Table 1-1 Agxt-qPCR-F / R) is detected Agxt D205N Rat liver Agxt mRNA level was found to significantly reduce ( Figure 4 -A). Consistent therewith, as compared to wild type control, in Western blot experiments, Agxt D205N Protein levels in rats almost invisible ( Figure 4 -B, Figure 4 -C), show D205N mutation greatly affect the actual level of AGT and stability, the height of which is consistent with the impact of humans most point mutations. AGT activity found in vitro assay, Agxt D205N AGT activity in rats was significantly lower than WT littermate rats ( Figure 4 -D).

[0081] (2) Characterization of PH1 rats urine:

[0082] We analyzed the urine, 24h urine reco...

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Abstract

The invention discloses a construction method, composition, kit and application of a disease model related to abnormal glyoxylic acid metabolism, and relates to the field of biotechnology. The construction method includes editing the Agxt gene of the target animal so that the encoded AGT protein has a D205N mutation. This construction method can obtain a new disease model related to abnormal glyoxylate metabolism. The disease model has high consistency of gene mutation and human gene mutation, early occurrence of high urinary oxalate, typical bladder stones formed spontaneously, and a single stone-induced disease model. The time for calcium oxalate and calcium deposition in the kidneys is shorter after drug induction, and the results obtained by using this model to study diseases related to abnormal glyoxylate metabolism, such as type Ⅰ primary hyperoxaluria or kidney stones, are more representative. more reliable.

Description

Technical field [0001] The present invention relates to the field of biotechnology, and in particular, there is a construction method, composition and kit and application of a acethemic metabolism-related disease model. Background technique [0002] Type I primary hypertational uremia (Primary Hyperoxaluria Type 1, PH1) is an AGXT gene mutation encoded by Alanine-Glyoxylate Aminotransferase, AGT, which has been discovered. More than 170 mutant types) caused rare normally syndrome. AGXT gene mutation results in damage to the AGT activity of the liver, the process of catalyzed by glyoxylic acid into glycine ( figure 1 ), Resulting in a large amount of acetalic acid in the liver to be oxidized into sore acids and almost all from the kidney, forming highly poliform regions, thereby causing urinary articularity, more than 50% of PH1 patients progressed as end-stage nephropathy. PHL patients often recurrent renal stones (calcium oxalate stones are deposited in renal pelvis, urethra), r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85
CPCA01K67/0278A01K2217/203A01K2227/105A01K2267/035
Inventor 耿红全郑锐李大力
Owner XIN HUA HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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