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Method for detecting pig hairless character based on ceRNA expression quantity and application of method

A technology for expression and quantitative detection, applied in DNA/RNA fragments, applications, recombinant DNA technology, etc., can solve problems such as affecting hair follicle growth, and achieve accurate results.

Inactive Publication Date: 2021-12-07
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is necessary to study the related genes and their expressions that affect the development of pig hair follicles, and there is no relevant research report at present
No studies have reported that BMPR1b, Sus-miR29a-5p and MSTRG.2162.1 can affect hair follicle growth through the regulation mechanism of ceRNA (MSTRG.2162.1 / Sus-miR29a-5p / BMPR1b), and this ceRNAs has not been used to detect pig The hairless trait of pigs, and there is no report on using ceRNA as a molecular marker to assist in the selection and breeding of hairless pigs

Method used

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  • Method for detecting pig hairless character based on ceRNA expression quantity and application of method
  • Method for detecting pig hairless character based on ceRNA expression quantity and application of method
  • Method for detecting pig hairless character based on ceRNA expression quantity and application of method

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1: Screening for ceRNAs associated with hairless traits

[0041] The experimental population of the present invention is a small amount of large white hairless pigs discovered earlier (discovery address: Anping County, Hengshui City, Hebei Province, discoverer: Ding Xiangdong, associate researcher of the Animal Science and Technology College of China Agricultural University)

[0042] Specific implementation of ceRNA screening:

[0043] S1. Hairless pig mating plan: select normal boars and hairless sows for mating, and perform cesarean section when pregnant sows reach 41 days of gestation, take out pig embryos and collect two tissue pieces of 2cm x 2cm on the back of the embryos, and then separate the skin tissue And disinfect. One of them was placed in liquid nitrogen for RNA preservation, and the other was placed in paraformaldehyde for later phenotypic identification.

[0044] S2. Phenotype identification: for phenotype identification, hematoxylin and eosin ...

Embodiment 2

[0055] Example 2. Verifying the relationship between the expression level of ceRNA (MSTRG.2162.1 / Sus-miR29a-5p / BMPR1b) and the hairless phenotype in pig embryonic skin

[0056] S1. Hairless pig breeding plan: select normal boars and hairless sows to breed, and perform cesarean section when pregnant sows reach 41 days of gestation, take out pig embryos and collect two tissue pieces of size 2cm x 2cm on the back of the embryos, then separate the skin tissue and dry Disinfect. Place samples in liquid nitrogen for RNA preservation.

[0057] S2. mRNA extraction of skin samples: Grind the tissue block into powder in liquid nitrogen, weigh 50-100 mg of the tissue sample, add 1 ml Trizol, and then perform homogenization. Using chloroform (trichloromethane), isopropanol, 75% ethanol, and RNase-free water (required to be prepared with DEPC-treated water), the protein, DNA, and inorganic salts in the tissue were eluted and removed, and the RNA precipitate was concentrated.

[0058] S3....

Embodiment 3

[0091] Example 3 Breeding Homozygous Hairless Pigs by Gene Editing BMPR1b Knockout Pigs

[0092] Hairless pigs were gene-edited using CRISPR / Cas9 gene knockout technology, and BMPR1b knockout was used to verify the molecular mechanism of BMPR1b on the formation of hair follicle placodes and the effect of hair follicle phenotype. And collected BMPR1b knock-in pig hair follicle phenotype data, analyzed and verified that BMPR1b is a functional gene that affects the formation of hair follicle placode and leads to hairless traits.

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Abstract

The invention relates to a method for detecting a pig hairless character based on a ceRNA expression quantity and application of the method. The method comprises the following steps: collecting a pig embryo back skin tissue with a gestation period of 41 days; quantitatively detecting the expression quantities of MSTRG.2162.1, sus-miR29a-5p or BMPR1b through PCR; and determining that a pig has no hair when the BMPR1b gene expression quantity is lower than 2, or the expression quantity of the sus-miR29a-5p is higher than 30, or the expression quantity of the MSTRG.2162.1 is lower than 10. The method for detecting the pig hairless character based on the ceRNA expression quantity, provided by the invention, can be used for assisting hairless pig screening and breeding; and a hairless pig homozygote can be cultivated and obtained through knocking out BMPR1b. According to the method provided by the invention, ceRNA influencing the growth of a hair follicle substrate is screened out and an important reference and an animal model are provided for researches of human hereditary alopecia diseases; and meanwhile, a molecular marker with the hairless character and a large white pig hairless test group which can be used for researches are also provided, and an important pig hairless strain animal model is provided for researches of diseases including the human hereditary alopecia, hairless diseases and the like.

Description

technical field [0001] The invention relates to the technical field of gene detection and breeding, in particular to a method for detecting the hairless trait of pigs based on ceRNA expression and its application. [0002] technical background [0003] Humans are very concerned about hereditary hair loss and hair thinning, and the stability of hair follicle morphology and function is the key to a normal hair cycle. The formation of hair originates from the morphological development of embryonic hair follicles, but the scarcity and ethics of human hair follicle samples, especially embryonic hair follicle samples, restrict the in-depth study of the morphology and developmental mechanism of embryonic hair follicle placodes. Pigs are of great significance in medicine. The genetic homology between pigs and humans is very high, and their genomes are similar to those of humans, with similar complexity and chromosomal composition. Therefore, as an ideal model animal for medical rese...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6851C12N15/113C12N15/12C12N15/85C12N15/877A01K67/027
CPCC12Q1/6888C12Q1/6851C12N15/1136C07K14/71C12N15/8509C12N15/8778A01K67/0276A01K67/0273C12Q2600/124C12Q2600/178C12Q2600/158C12N2800/107C12N2310/141A01K2217/075A01K2217/15A01K2227/108A01K2267/03C12Q2531/113C12Q2525/207C12Q2563/107C12Q2545/113
Inventor 丁向东蒋尧刘华涛邹全李淑娟
Owner CHINA AGRI UNIV