Single cell cloning method for obtaining goat mammary epithetical cells

A breast epithelial cell and single cell cloning technology, which is applied in the direction of animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problem of low cell clone formation rate and achieve the effect of improving the single cell clone formation rate

Active Publication Date: 2015-02-25
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, to obtain single-cell clonal populations by limiting dilution or other methods, cells must face the disadvantage of growing at very low densities.
Most studies have shown that the colony formation rate of cells is very low under very low density conditions

Method used

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  • Single cell cloning method for obtaining goat mammary epithetical cells
  • Single cell cloning method for obtaining goat mammary epithetical cells
  • Single cell cloning method for obtaining goat mammary epithetical cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Sampling and Processing of Goat Mammary Epithelial Cells

[0060] The mammary gland tissues of the purebred Laoshan dairy goats of Qingdao Aotai sheep farm in the lactation period of 30 days were directly aseptically collected by using the existing technology;

[0061] Rinse the collected breast tissue block with PBS to remove the blood and goat milk on its surface; after rinsing, immerse it in DF12 medium containing 200U / mL penicillin and 200μg / mL streptomycin, put it in an ice box and quickly bring it back to the laboratory For treatment, the breast tissue was washed repeatedly with PBS containing 200 U / mL penicillin and 200 μg / mL streptomycin until the washing liquid became clear, divided and trimmed on an ultra-clean bench, and the adipose tissue and connective tissue were removed and rinsed until the washing liquid was clear. White granular acinar tissue can be obtained.

Embodiment 2

[0062] Example 2 Tissue Block Culture Isolation of Mammary Epithelial Cells and Fibroblasts

[0063] a. Use ophthalmic scissors and ophthalmic forceps to divide the above-mentioned white granular acinar tissue into 0.5-1mm 3 The left and right small tissue pieces were rinsed with PBS containing 200 U / mL penicillin and 200 μg / mL streptomycin;

[0064] b. Infiltrate the small tissue pieces with the medium prepared by FBS and DF12 at a volume ratio of 1:1 for 2-4 minutes, inoculate them on the bottom of the culture dish at an interval of 0.5 cm, and place them at 37°C and 5vt% CO 2 , in an incubator with saturated humidity for 4 hours;

[0065] c. Add culture medium to the above-mentioned petri dish so that it just covers the bottom of the petri dish, and place at 37°C, 5vt% concentration of CO 2 , cultured in an incubator with saturated humidity, add culture solution after 12 hours until the tissue block is completely submerged, replace half of the culture solution after 2 day...

Embodiment 3

[0069] Fibroblasts were removed by rapid digestion combined with differential attachment method to obtain purified mammary epithelial cells. The specific steps are as follows:

[0070] a. Differential digestion method: add digestive fluid to the culture of breast epithelial cells grown with other cells to digest the cells, observe the changes in cell morphology under an inverted microscope, and when the fibroblasts are detached from the bottom wall of the culture dish, aspirate and digest Remove the fibroblasts, use the remaining digestion solution to continue digestion for 1-2 minutes, add culture medium to stop the digestion, and pipette the remaining cells into a single-cell suspension;

[0071] Wherein the digestion solution is an equal volume of PBS without calcium and magnesium ions and 0.25% Trypsin-EDTA solution, the digestion condition is room temperature 25°C, and the digestion time of fibroblasts is 1.5-2 minutes;

[0072] The culture solution is a solution with a v...

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Abstract

The invention relates to the fields of tissue and cell engineering and particularly provides a single cell cloning method for obtaining goat mammary epithetical cells. According to the method, single cell cloning preparation is performed through a micromanipulation method, tedious cell counting and repeated dilution in a traditional method are eliminated, single cell cloning operation can be accomplished within a short time, and the single cell cloning formation rate is increased. The goat mammary epithetical cells obtained through the method provided by the invention can be used for researching functions of different lineages of mammary epithelial cells and the interaction of the mammary epithelial cells during a lactation process.

Description

technical field [0001] The invention relates to the field of tissue and cell engineering, and in particular provides a method for obtaining single-cell clones of goat mammary gland epithelial cells. Background technique [0002] Mammary gland epithelial cells are an in vitro model for studying mammary gland growth and development, lactation mechanism, and verifying the effectiveness of mammary gland tissue-specific expression vectors. The mature mammary parenchyma contains three types of cells, the ductal epithelium, the secretory epithelium, and the myoepithelium. In recent years, researchers have established a large number of cell lines using mammary epithelial cells as models, but most of the existing studies cannot properly distinguish the functions of cells of each lineage and their interactions during lactation. [0003] The methods for obtaining primary mammary epithelial cells mainly include enzymatic digestion, milk separation, and tissue block culture. No matter ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
Inventor 王建民陈存仙董飞王桂芝张赛赛秦孜娟
Owner SHANDONG AGRICULTURAL UNIVERSITY
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