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Building method of secreting type Laoshan dairy goat mammary gland epithelial cell line

A technology of mammary epithelial cells and construction methods, which is applied in the field of Laoshan dairy goat mammary epithelial cell line construction, can solve the problem of not many mammary epithelial cell lines being reported, and achieve the effect of strong growth potential

Active Publication Date: 2020-05-15
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, there are not many reports on animal mammary epithelial cell lines

Method used

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  • Building method of secreting type Laoshan dairy goat mammary gland epithelial cell line
  • Building method of secreting type Laoshan dairy goat mammary gland epithelial cell line
  • Building method of secreting type Laoshan dairy goat mammary gland epithelial cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Construction of Laoshan Dairy Goat Mammary Epithelial Cell Line

[0046] 1.1 Culture of goat mammary epithelial cells

[0047] Healthy Laoshan dairy goat mammary gland tissues on the 35th day of lactation were taken during aseptic surgery, soaked in 75% alcohol for disinfection, rinsed with PBS to remove adipose tissue and connective tissue, and then rinsed with PBS until the solution was no longer turbid. Then cut up the acinar tissue as much as possible and wash it clean, put it into a centrifuge tube, add 0.2% collagenase Ⅰ three times the volume of the chopped acinar tissue, digest it with shaking at 37°C until the tissue pieces are blurred, add Dilute with an appropriate amount of PBS, blow the dispersed cells through a 200-mesh cell sieve, collect the filtrate, centrifuge at 1500rpm for 10 minutes, discard the supernatant, add an appropriate amount of culture medium to resuspend the cells, and the culture medium contains 10% fetal bovine serum, 5 μg / m...

Embodiment 2

[0059] Example 2: Verification of characteristics of primary goat mammary epithelial cells and constructed cell lines

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Abstract

The invention discloses a building method of a secreting type Laoshan dairy goat mammary gland epithelial cell line. The building method disclosed by the invention comprises the following steps: transfecting an exogenous expression vector containing a target gene SV40LT to dairy goat mammary gland epithelial cells to build the transgenic dairy goat mammary gland epithelial cell line; observing anexpression condition of a maker gene copGFP through a fluorescence microscope; and carrying out Puromycin screening to obtain positive cells, wherein PCR verification proves that the target gene SV40LT is integrated into a gene group of the dairy goat mammary gland epithelial cells. According to the building method disclosed by the invention, the built Laoshan dairy goat mammary gland epithelial cell line has an unlimited passage function, can maintain the characteristics of the dairy goat mammary gland epithelial cells, and has functions of synthesizing and secreting proteins, and in the passage process, a core type can be maintained to be normal, and the change of a chromosome structure and the quantity is avoided; and moreover, the Laoshan dairy goat mammary gland epithelial cell line has susceptibility to Gram-negative bacteria, can be used for building animal models having inflammatory response and can be used for researching related diseases.

Description

technical field [0001] The invention relates to the field of transgenic technology, in particular to a method for constructing a secretory Laoshan dairy goat mammary gland epithelial cell line. Background technique [0002] Mastitis is a common disease in the feeding and management of small ruminants. It is the most common in dairy farming. In recent years, with the continuous discovery of the value of goat milk, the dairy goat breeding industry has also developed rapidly, but the problem of mastitis has also become increasingly prominent, causing serious losses to the dairy goat breeding industry. First of all, mastitis has caused serious economic losses to the dairy goat breeding industry, and secondly, dairy goat udders also have an impact on the quality of milk and its products. In addition, mastitis, like dairy cows, will greatly shorten the economical use of dairy goats, and mastitis of dairy goats also poses a potential biosafety risk to consumers. Although antibiot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10C07K14/47C12P21/02
CPCC12N15/86C12N5/0631C07K14/4732C12N2740/15043C12N2510/02
Inventor 苏峰张鸿焱葛利江姜运良李伯玉刘营
Owner SHANDONG AGRICULTURAL UNIVERSITY
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