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Construction method of spontaneous kidney yin deficiency Ptgds gene knockout rat model

A gene knockout, rat model technology, applied in the field of bioengineering, can solve the problems of long time and difficulty in feeding model animals, and achieve the effect of targeting specificity and high modification accuracy.

Pending Publication Date: 2022-06-24
SHAANXI UNIV OF CHINESE MEDICINE
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Problems solved by technology

[0005] In order to conduct related research on menopausal kidney yin deficiency, the bilateral ovaries of female rats are generally surgically removed to prepare animal models. , adrenaline and ovary and other endocrine experiments; however, the construction of rat models has the following problems: it takes too long, and it is difficult to raise model animals, so there is an urgent need for an efficient and stable method to construct animal models. In order to solve this problem, but the prior art only has the report of the Ptgds gene knockout (Ptgds- / -) mouse model, there is no relevant report about the rat Ptgds- / - knockout model

Method used

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  • Construction method of spontaneous kidney yin deficiency Ptgds gene knockout rat model
  • Construction method of spontaneous kidney yin deficiency Ptgds gene knockout rat model
  • Construction method of spontaneous kidney yin deficiency Ptgds gene knockout rat model

Examples

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Embodiment

[0070] The following describes the construction method of the rat model with a specific example.

[0071] 1. Ptgds gene information of SD rats

[0072] The rat Ptgds gene constructed in this example is located on chromosome 3 of SD rats, and there is only one transcript: Ptgds-201.

[0073] Therefore, this example builds a model with the Ptgds-201 transcript as the object.

[0074] 2. SD rat Ptgds protein information

[0075] see figure 2 and image 3 , the SD rat Ptgds protein domain and the SD rat Ptgds protein expression profile provided in this example.

[0076] 3. Construction ideas of rat knockout model

[0077] The specific sgRNA (single-guide RNA) of the rat Ptgds gene was used to mediate the cleavage of DNA by Cas9 nuclease to produce specific DSBs (Double-Stranded Breaks), and the entire coding sequence of the gene was deleted,

[0078] see figure 1 , when implemented, nicks are made on exon 1 of the spliceosome Ptgds-201 and the non-coding region of exon 7, ...

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Abstract

The invention belongs to the technical field of bioengineering, and relates to a construction method of a spontaneous kidney yin deficiency Ptgds gene knockout rat model, which comprises the following steps: 1) designing a target sequence Ptgds-sgRNA1 / 2; (2) purifying Cas9 mRNA (messenger ribonucleic acid) and Ptgds-sgRNA1 / 2; (3) carrying out targeted knockout on the sequence fragments in the Ptgds gene by utilizing a CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / associated protein 9) system; (4) injecting the purified Cas9mRNA, the purified Ptgds-sgRNA1 / 2 and the purified Ptgds knockout gene into a rat embryo to obtain a newborn rat; (5) identifying and selecting heterozygous rats; and 6) further performing multi-generation breeding with a wild rat to obtain the Ptgds gene knockout rat model. The method has the advantages of being high in gene modification accuracy, specific in targeting and short in experimental period, a reliable and stable gene engineering model is developed, and a foundation is laid for the treatment effect of climacteric syndrome related diseases.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a construction method of a spontaneous kidney-yin deficiency Ptgds gene knockout rat model. Background technique [0002] Perimenopause generally refers to the natural menopausal state after the depletion of follicles in a woman's ovaries and the emergence of various symptom phenotypes around the "brain-kidney-reproductive" axis, such as hot flashes (or vasomotor symptoms), chronic kidney disease , cardiovascular disease, neurodegenerative disease, etc. These physical or mental symptoms are called perimenopausal syndrome. Long-term studies have shown that perimenopausal syndrome is closely related to the development of chronic kidney disease. From the perspective of TCM dialectics, perimenopausal syndromes are based on kidney deficiency, mostly kidney yin deficiency, typically hot flashes, obesity, Central degenerative symptoms are mainly due to women entering around 49 yea...

Claims

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Application Information

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IPC IPC(8): C12N15/873A01K67/027C12N15/61
CPCC12N15/873A01K67/0276C12N9/90C12Y503/99002A01K2227/105A01K2267/03C12Q1/6888A01K2217/075A01K2267/035C12N15/907C12N9/22
Inventor 刘妍如唐志书宋忠兴
Owner SHAANXI UNIV OF CHINESE MEDICINE
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