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A method for isolation and cultivation of PGCs derived from chicken reproductive crescent

A technology of separation and culture and crescent is applied in the field of separation and culture of chicken reproductive crescent-derived PGCs, which can solve the problems of poor repeatability, limit the research of PGCs, need to be improved, etc., and achieve the effects of simple operation, good proliferation activity, and good repeatability.

Active Publication Date: 2018-08-24
FOSHAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Different laboratories use different isolation methods and culture systems for PGCs. The mechanism of PGCs self-renewal and pluripotency maintenance in vitro is not yet clear, and the isolation operation of chicken embryo blastoderm is complicated and has poor reproducibility, which limits further research on PGCs.
Therefore, the current separation technology and culture system of chicken PGCs still need to be improved.

Method used

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  • A method for isolation and cultivation of PGCs derived from chicken reproductive crescent
  • A method for isolation and cultivation of PGCs derived from chicken reproductive crescent
  • A method for isolation and cultivation of PGCs derived from chicken reproductive crescent

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Embodiment Construction

[0076] The preferred embodiments of the present invention will be described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention.

[0077] refer to Figure 1 to Figure 3 :

[0078] I material

[0079] The microporous membrane used in the following examples is the product of Shanghai Xingya Company;

[0080] Fresh eggs (ephedra chicken) were purchased from Nanhai Breeding Poultry Co., Ltd., Foshan City, Guangdong Province;

[0081] Trypsin, fetal bovine serum and chicken serum were all purchased from HyClone Company;

[0082] SCF, LIF, and bFGF cytokines were all purchased from PeproTech;

[0083] RNA extraction kit, DNA Marker, AMV reverse transcription kit, and Ex-Taq enzyme were all purchased from TaKaRa Company;

[0084] Anti-SSEA-1 antibody was purchased from SANTA CRUZ company;

[008...

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Abstract

The invention relates to a separation culture method for chick germinal crescent source PGCs. The method comprises the following steps that firstly, according to a separation method, a clean chick embryo without yolk contamination is extracted from a hatching egg through a filter film pasting method, and then germinal crescent tissue is obtained from the chick embryo; secondly, the germinal crescent tissue is digested into single cells through pancreatin, and purification is carried out on the PGCs in the single cells; thirdly, the purified PGCs are placed in a culture system including a BRL-3A cell feed layer and a PGCs complete culture solution to be cultured. The PGCs come from the chick embryo germinal crescent zone, the PGCs content of the 5-8 HH period chick embryo germinal crescent zone is high, better proliferation vitality is achieved, and high-level pluripotency is maintained. The chick embryo germinal disc is separated through the filter film pasting method, operation is easy, repeatability is good, the clean chick embryo without yolk contamination is separated from the hatching egg, and the purified PGCs are obtained. The PGCs cultured through separation can be proliferated to the maximum for later experiment operation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for separating and culturing PGCs derived from chicken reproductive crescent. Background technique [0002] Stem cells are a kind of early undifferentiated cells with differentiation potential and self-renewal ability. According to different differentiation potentials, stem cells can be divided into totipotent stem cells, pluripotent stem cells and unipotent stem cells. Chicken Primordial Germ Cells (PGCs) are the progenitor cells of all levels of germ cells and the precursor cells that can develop into sex cells (sperm and oocyte). Under appropriate culture conditions in vitro, its morphology, surface markers and differentiation potential are similar to embryonic stem cells (EmbryonicStem Cells, ESCs). As reproductive stem cells, PGCs can not only be used to study the development and differentiation of germ cells, but also can be used as an effective carrier f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0735
Inventor 王丙云陈志胜陈胜锋计慧琴李东升冼琼珍
Owner FOSHAN UNIVERSITY
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