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Hyperuricemia rat model and construction method thereof

A technology of hyperuricemia and rat model, applied in the field of genetic engineering, can solve problems such as intestinal barrier damage and kidney damage, and achieve the effect of good reactivity and good application prospect.

Inactive Publication Date: 2020-07-17
段为钢
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Qingdao University subsequently used CRISPR / Cas9 technology to regain uricase-deficient mice, but the above defects have not been well resolved, such as severe kidney damage, severe intestinal barrier damage, etc.

Method used

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  • Hyperuricemia rat model and construction method thereof
  • Hyperuricemia rat model and construction method thereof
  • Hyperuricemia rat model and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of uricase gene knockout heterozygous rats

[0039] The rat uricase (Uox) gene is located on chromosome 2 (ENSRNOG00000016339), and the full length of the gene is 36.13kb. The gene has 8 exons in total. The ATG start sequence is located in exon 1, and the TGA termination sequence is located in exon 8. The transcribed mRNA has a total of 1359bp, encoding 303 amino acid residues. Exon 2-4 encodes the catalytic active center of uricase, so this sequence was selected as the target region for targeting.

[0040] Construct a set of gRNA sequences, which recognize the 11506-17370 base sequence of the Uox gene, covering the exon 2-4 region, which is also the target region for deletion. The gRNA was obtained by amplifying the rat genome with primers.

[0041] The specific primer sequences are as follows:

[0042] 5'-CCACTAGGCTAGGCGTAGCAAGG-3'; SEQ ID NO.1;

[0043] 5'-TTTTCATATTGACTGACGGCAGG-3'; SEQ ID NO.2.

[0044] Ligate the obtained gRNA to the CRI...

Embodiment 2

[0058] Example 2 Preparation of uricase-deficient homozygous animals

[0059] Female and male Uox+ / - rats (F0) were obtained by normal feeding, and the next generation of Uox- / - rats (F1) were obtained by mating the rats after sexual maturity. First, take blood from the tail vein of F1 rats and use tungsten blue method to detect the serum uric acid level, and screen possible Uox- / - rats (F1) from the F1 rats according to the serum uric acid level. The male serum uric acid is higher than 40 μg / ml, and the female Higher than 30 μg / ml is a possible Uox- / - rat (F1). The possible Uox- / - rats (F1) were mated with males and females, and after giving birth to offspring (F2), the parental rats (F2) were identified by liver enzyme activity, uricase Western Blot and liver uricase mRNA PCR. It was confirmed from multiple levels that the possible parental Uox- / -rats did not express uricase.

[0060] Uox- / -rat liver homogenate uricase activity identification results see figure 1 , figur...

Embodiment 3

[0066] Breeding of embodiment 3 hyperuricemia rats

[0067]Male Uox- / - F2 rats with serum uric acid levels higher than 40 μg / ml and female serum uric acid levels higher than 30 μg / ml were reared and mated under normal diet conditions to obtain Uox- / - F3 rats. The obtained F3 rats have a relatively large base, and further select male rats with serum uric acid higher than 50 μg / ml and females with serum uric acid higher than 40 μg / ml for breeding and mating under normal dietary conditions to produce Uox- / -F4 rats . Among the F4 rats, male rats with serum uric acid higher than 60 μg / ml and female rats with serum uric acid higher than 50 μg / ml were selected for breeding and mating under normal diet conditions to produce Uox- / -F5 rats. Among the F5 rats, male rats with serum uric acid higher than 70 μg / ml and female rats with serum uric acid higher than 60 μg / ml were selected for breeding and mating under normal diet conditions to produce Uox- / -F6 rats. After that, continue to se...

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Abstract

The invention discloses a hyperuricemia rat model and a construction method thereof, and belongs to the technical field of genetic engineering. According to the hyperuricemia rat model disclosed by the invention, firstly, a CRISPR / Cas9 technique is adopted for knocking out uricase genes of SD rats to obtain heterozygote, then through a serum uric acid phenotype screening technique, a liver homogenate enzymatic activity technique, a liver RNART-PCR technique and a liver homogenization protein WesternBlot technique, free uricase expression is proved, and further uricase deletion rates (Uox- / -rat) are obtained; and then, through repeated seed selection and hybridization, the rats are bred for 7 generations, and the serum uric acid level of male and female rates can achieve a diagnosis level.The survival time of the model within 1 year can achieve 95%, and the model has generativity. The hyperuricemia rat model is particularly suitable for research on a hyperuricaemia mechanism, uric acidreducing medicines are screened, and the model can be further used for research of gout and has favorable application prospects.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a hyperuricemia rat model and a construction method thereof. Background technique [0002] Gout (gout) is a common and frequently-occurring disease in modern society, and it occurs more frequently in men. The main pathogenesis is related to long-term purine metabolism disorders and increased serum uric acid. It is generally believed that hyperuricemia (hyperuricemia, higher than 70 μg / ml in men, ie 420 μM) is the basis of gout. The disease has a long history, and it was more common in Europe and North America. After the 1980s in my country, the prevalence of the disease has a clear upward trend, and men are higher than women. For example, the epidemiological survey in Shandong coastal areas in 2006 showed that the prevalence of hyperuricemia in men was 18.3%, and that of gout was 1.94%. %. There is no large-scale epidemiological survey at present, but it is reported...

Claims

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Application Information

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IPC IPC(8): A01K67/027
CPCA01K67/0275A01K2217/075A01K2227/105A01K2267/0368
Inventor 段为钢
Owner 段为钢
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