Method for detecting pig hair-free character based on EDAR gene expression quantity and application thereof

A gene expression and expression technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of unassisted hairless pig selection and breeding, etc., to reduce the existence of false positives and achieve accurate results Effect

Pending Publication Date: 2021-11-23
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on using EDAR to detect the hairless traits of pigs, and there is n...

Method used

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  • Method for detecting pig hair-free character based on EDAR gene expression quantity and application thereof
  • Method for detecting pig hair-free character based on EDAR gene expression quantity and application thereof
  • Method for detecting pig hair-free character based on EDAR gene expression quantity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Screening for the gene EDAR associated with hairless traits

[0040] 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)

[0041] The specific implementation of EDAR screening:

[0042] 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.

[0043] S2. Phenotype identification: for phenotype identification, hematoxylin ...

Embodiment 2

[0058] Example 2 Verification of the relationship between EDAR gene expression and hairless phenotype in pig skin (experimental verification of the relationship between EDAR quantitative values ​​and hairless traits)

[0059] 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.

[0060] 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 th...

Embodiment 3

[0085] Example 3 Breeding Homozygous Hairless Pigs by Gene Editing EDAR Knockout Pigs

[0086] Hairless pigs were gene-edited using CRISPR / Cas9 gene knockout technology, and the molecular mechanism of EDAR on the formation of hair follicle placodes and the effect of hair follicle phenotype were verified by knockout of EDAR. The phenotype data of EDAR knock-in pig hair follicles were collected and analyzed to verify that EDAR is a functional gene that affects the formation of hair follicle placodes and leads to hairless traits.

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Abstract

The invention relates to a method for detecting pig hair-free character based on EDAR gene expression quantity and application thereof, back skin tissue of a pig embryo is collected, the EDAR gene expression quantity is quantitatively detected through PCR, and the pig with the EDAR gene expression quantity lower than 3 is judged as a hair-free pig. The method for detecting the pig hair-free character based on the EDAR gene expression quantity can be used for assisting screening and breeding of hair-free pigs, and hair-free pig homozygotes can be obtained through breeding by knocking out EDAR. The candidate gene EDAR influencing the growth of the hair follicle substrate is screened out, an important reference and an animal model are provided for research of human hereditary alopecia diseases, meanwhile, a molecular marker with the hairless character and a large white pig hairless test group capable of being used for research are also provided, and an important pig hair-free strain animal model is provided for research on human hereditary alopecia, hair-free and other diseases.

Description

technical field [0001] The invention relates to the technical field of gene detection and breeding, in particular to a method for detecting hairless traits of pigs based on EDAR gene expression and an application thereof. [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 medi...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/158C12Q2600/124
Inventor 丁向东蒋尧刘华涛邹全李淑娟
Owner CHINA AGRI UNIV
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