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Method of efficiently separating embryonic stem cells of poultry

A technology of embryonic stem cells and blastodisc cells, which is applied to embryonic cells, germ cells, animal cells, etc., can solve the problems of cumbersome operation, low survival rate, and cumbersome and complicated methods of hair ring method, and achieve simple and easy methods and improve survival High rate, repeatable effect

Inactive Publication Date: 2013-04-03
CHINA AGRI UNIV
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Problems solved by technology

The paper ring method tears the blastoderm when obtaining the blastoderm, resulting in the separation of epiblast and hypoblast, resulting in the loss of a large number of epiblast embryonic stem cells when the preformed solution is washed; Long; the medicine spoon method is easy to introduce a large amount of yolk, and it is not easy to separate the blastoderm, and these methods all need to prepare or prepare small objects, wash the blastoderm in the prefabricated solution at 37 degrees, and it is easy to lose a large number of blastoderm cells during this process , in this process, due to the stimulation of temperature, premature cell recovery is activated, and it is easy to cause stress death of embryonic stem cells in multiple washings
[0007] Existing methods use trypsinization to digest stem cells into single cells and place them on feeder cells for isolation and culture. This is not in line with the development trend of stem cell culture, because trypsin has a strong digestion effect on cells, and trypsinization into single cell culture It is easy to cause anoikis in stem cells, and long-term use of trypsin will increase the possibility of chemical aberrations in stem cells, resulting in loss or damage of chromosomes
[0008] In terms of culture medium, the existing method adopts chicken embryo fibroblast feeder layer for adherent culture and adds 10% calf serum and 2% chicken serum to the medium for culture. Chicken embryo fibroblast feeder layer needs to be prepared in advance and is easy to introduce The influence of cells with different genetic backgrounds, due to the differences in batches of chicken embryo fibroblasts, the serum contains a variety of unknown components, and the hormone molecules contained in it, such as estrogen (P4), easily lead to the differentiation of stem cells, so that the established The embryonic stem cell line is doped with various unknown and uncontrollable factors, which reduces the reliability and repeatability of the experiment, which is not conducive to subsequent research and the preparation of transgenic animals
[0009] The existing method of separating and preparing poultry stem cells is cumbersome and complicated, with low efficiency and low survival rate. It needs to consume several eggs with different genetic genes to establish, and there are huge doubts about reliability and repeatability, and it is impossible to quantify and accurately control the quality. Therefore, it is particularly important to establish an efficient method for isolating avian embryonic stem cells

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  • Method of efficiently separating embryonic stem cells of poultry
  • Method of efficiently separating embryonic stem cells of poultry

Examples

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Embodiment 1

[0049] Example 1 Obtain all blastodisc cells from a chicken egg that has just been born, digest and clean them, and carry out primary culture

[0050] 1. Lay the breeding eggs of chickens that have been cleaned and disinfected with bromogeramine and stored at 2-6°C for 0-2 days after production, and placed in a low-temperature freezer overnight;

[0051] 2. Keep the laying eggs horizontally, move them into the ultra-clean workbench, and disinfect with alcohol cotton balls at the highest point of the equatorial surface of the eggs and a radius of 2-3cm around them;

[0052] 3. Use sterile ophthalmic tweezers to open a window with a radius of 2 cm at the highest point of the equatorial surface of the laying eggs, and you can see the position of the germinal disc directly below it;

[0053] 4. Use ophthalmic scissors to cut the vitelline membrane around the blastodisc;

[0054] 5. Use a pipette gun equipped with a 1ml sterilized tip with a cut-off tip of about 0.5-1cm to aim at ...

Embodiment 2

[0063] Example 2 Enrichment and Purification of Embryonic Stem Cells from Primary Cultured Blastodisc Cells

[0064] 1. Take out the culture dish after primary culture for 24 hours, tilt it at 30°, and use a 5ml pipette to collect the culture medium;

[0065] 2. Filter the medium through a sterilized cell sieve with a mesh opening size of 40 μm to remove dead cells and blastoderm hypoblast cells;

[0066] 3. Invert the cell sieve, place it on another Petri dish, and elute with 5ml of fresh serum-free mTeSR1 medium containing Y-27632 at a final concentration of 10μM / ml to obtain a large amount of enriched and purified Embryonic stem cells from the blastoderm epiblast.

[0067] There may be a small amount of vitelline membrane fragments remaining in the above process, which can be removed with a pipette gun under a stereoscopic microscope; the enriched and purified chicken embryonic stem cells can be used for subsequent gene transfection operations, and can be used to prepare t...

Embodiment 3

[0068] Example 3 The isolated and cultured primary chicken embryonic stem cells were identified by SSEA-1 and DAPI staining

[0069] The separated and purified chicken embryonic stem cell pellets were transferred to ordinary cell culture dishes, and their morphology was observed the next day (24 hours) after their adherent culture.

[0070] 1. Fix the adherent embryonic stem cell samples in 4% paraformaldehyde for 10 minutes, wash with PBS twice, 5 minutes each time;

[0071] 2. Add 0.3% Ttiton-X1002mL, place at room temperature for 10min, then wash twice with PBS, 5min each time;

[0072] 3. The sample was blocked in 5% BSA for 20 minutes, and the primary antibody (mouse anti-SSEA-1 IgM) was diluted with PBS at a dilution ratio of 1:500. The sample was incubated with the primary antibody for 1 hour at room temperature, and then washed twice with PBS, 5 minutes each time;

[0073] 4. The secondary antibody used is biotinylated goat anti-mouse IgM, diluted 1:100 with PBS, incu...

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Abstract

The invention relates to a method of efficiently separating embryonic stem cells of poultry. The method comprises the steps: 1) taking out an embryonic disc from a hatching egg; 2) digesting and cleaning the embryonic disc and removing yolk of the embryonic disc; 3) conducting primary culture to the cells of the digested embryonic disc; and 4) enriching and purifying the embryonic stem cells from the cells of the primarily cultured embryonic disc. The method is simple and easy, accurate and effective, high in repeatability and low in cost, equipment or reagent in the whole separation and culture system can be self-made or commercialized, a feeding layer is not prepared, enzymatic digestion and related processing are not needed, blood serum is not required, and about 20 thousand purified embryonic stem cells can be separated and cultured from one hatching egg, and can be used for in vitro transfection to conduct gene modification or apparent modification, thereby compacting the technological base for the transgenic poultry, installing a boost accelerator for development of a bioreactor and the realization of huge commercialization value, and having great scientific research values and popularization significance.

Description

technical field [0001] The invention relates to the technical field of separation of embryonic stem cells, in particular to a method for efficiently separating avian embryonic stem cells. Background technique [0002] Embryonic stem cells are the original seed cells that can proliferate for a long time without differentiation, and can differentiate into a variety of adult functional cells under certain conditions. Embryonic stem cells can proliferate rapidly under suitable growth conditions. The doubling time of human embryonic stem cells is about 24 hours, and the doubling time of mouse embryonic stem cells is faster, generally less than 24 hours. In recent years, the technology of mammalian embryonic stem cells represented by humans has developed rapidly, and its culture system has gradually changed from a culture system containing serum non-quantitatively combined with a feeder layer to a fully quantitative feeder-free culture system without serum, and the cultivation of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735
Inventor 李赞东蒋斌燕丽张文新万志毅
Owner CHINA AGRI UNIV
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