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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  • Claims
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Problems solved by technology

Qingdao University subsequently used CRISPR/Cas9 technology to regain uricase-deficient mice, but the abov

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

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

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

[0040] A set of gRNA sequences were constructed, which identified the 11506-17370 nucleotide sequence of the Uox gene, covering the exon 2-4 region, which was also the target region for deletion. Use primers to amplify the rat genome to obtain gRNA.

[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] The obtained gRNA was liga...

Example Embodiment

[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) could be obtained by mating after the rats were sexually mature. First, blood was collected from the tail vein of the F1 rat to detect the serum uric acid level by tungsten blue method. According to the serum uric acid level, the possible Uox- / - rats (F1) were screened from the F1 rat. Higher than 30 [mu]g / ml are possible Uox- / - rats (F1). The possible Uox- / - rats (F1) were mated with males and females, and the offspring (F2) were produced, and then the parental rats (F2) were identified by liver enzyme activity, uricase Western Blot identification and liver uricase mRNA PCR identification. It was confirmed from multiple levels that the parental possible Uox- / - rats did not express uricase.

[0060] The results of identification of uricase activity in Uox- / - rat liver homogenate are shown i...

Example Embodiment

[0066] Example 3 Breeding of hyperuricemia rats

[0067]Uox- / -F3 rats were obtained by feeding and mating male Uox- / - serum uric acid higher than 40μg / ml and female serum uric acid level higher than 30μg / ml under normal diet conditions. The obtained F3 rats have a relatively large base, and the male serum uric acid higher than 50μg / ml and the female serum uric acid higher than 40μg / ml were further selected for feeding and mating under conventional diet conditions to produce Uox- / -F4 rats . In F4 rats, males with serum uric acid higher than 60μg / ml and females with serum uric acid higher than 50μg / ml were selected for feeding and mating under conventional diet conditions to produce Uox- / -F5 rats. In F5 rats, males with serum uric acid higher than 70μg / ml and females with serum uric acid higher than 60μg / ml were selected for feeding and mating under conventional diet conditions to produce Uox- / -F6 rats. After that, continue to select male rats with serum uric acid higher than ...

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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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