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Avian pluripotent embryonic germ cell line

A cell line and germ cell technology, applied in the field of avian pluripotent embryonic germ cells, can solve the problems of no instruction or expected continuous proliferation, no investigation of other characteristics, and inability to determine EG cells.

Inactive Publication Date: 2002-10-09
韩在容 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, only the alkaline phosphatase activity of EG cells was examined to demonstrate their pluripotency, but not their other characteristics, so it was not possible to determine whether EG cells were actually established
In addition, the reference does not teach or predict whether EG cells have in vitro and in vivo differentiation capacity, which is a property of established EG cells, nor whether they proliferate continuously after several passages

Method used

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  • Avian pluripotent embryonic germ cell line
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  • Avian pluripotent embryonic germ cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Additionally, the percentages given below for solid-in-solid mixtures, liquid-in-liquid, and solid-in-liquid are on a wt / wt, vol / vol and wt / vol basis, respectively, unless specifically stated otherwise. Example 1: Isolation of PGCs and establishment of culture conditions for preparing chicken EG cell lines (step 1) Isolation of PGCs

[0040] Fertilized eggs of Bailaihang chickens obtained from the College of Agriculture and Life Sciences, Seoul National University were incubated at 37.5° C. and a relative humidity of 60-70% for 5.5 days (until the 28th stage). Embryos were extracted from fertilized eggs at stage 28 and washed with magnesium-free phosphate-buffered saline (PBS) in a 100 mm Petri dish to remove yolk and blood. Transfer the embryos to a black wax-coated Petri dish from which the embryonic gonads are isolated using forceps. Gonadal tissue was treated with 0.25% trypsin-0.05% EDTA to dissociate it into individual gonadal primordial germ cells (gPGC). They ...

Embodiment 2

[0042] Experiments have shown that EG cells will not colonize in the absence of IL-11 and IGF-1. Therefore, IL-11 and IGF-I are critical for the survival and proliferation of chicken EG cells. Embodiment 2: the culture of chicken EG cell

[0043] The EG cells obtained in Example 1 were inoculated in DMEM (Gibco, USA) supplemented with 10% FBS, 2% chicken serum (Gibco, USA), 1 mM sodium pyruvate, 2 mM L-glutamine, 5.5 × 10 -5 M β-mercaptoethanol, 100 μg / ml streptomycin, 100 units / ml penicillin, 5 ng / ml human stem cell factor (hSCF; Sigma, USA), 10 units / ml murine leukemia inhibitory factor (mLIF; Sigma, USA), 10 ng / ml bovine basic fibroblast growth factor (bFGF; Sigma, USA), 0.04ng / ml human interleukin-11 (h-IL-11; Sigma, USA), and 10ng / ml human insulin-like-growth factor-I (IGF-I; Sigma, USA) in a 24-well culture plate of EG cell culture medium, at 37 ° C, containing 5% CO 2 Culture in an atmospheric incubator for 7-10 days to prepare EG cell colonies deposited on the germ...

Embodiment 3

[0044] Figure 1a and 1b Chicken EG cell colonies after 3 passages are shown on chicken embryonic fibroblasts (CEF) ( Figure 1a : ruler, 50 μm; Figure 1b : scale bar, 25 μm). The morphology of chicken EG cells is slightly different from that of mouse ES living EG cells. Almost all chicken EG cell colonies were circular without firm borders with the CEG feeder layer. In contrast to mouse ES or EG cells, chicken EG cells do not pack tightly together; therefore individual member cells are not difficult to distinguish. The morphology of the colonies is multilayered with well-defined boundaries. Chicken EG cells have a large nucleus and a relatively small amount of cytoplasm, but their nucleoli are inconspicuous. Example 3: Characteristics of EG cells

[0045] To determine whether the pluripotency of EG cells has characteristic properties of pluripotent cells, the presence of glycogen and SSEA-1 epitopes, their alkaline phosphatase activity and their ability to proliferate a...

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Abstract

Disclosed in this invention is a process for preparing an established avian embryonic germ cell line comprising the steps of: (a) culturing primordial germ cells (PGCs) isolated from an avian embryonic gonad in a medium supplemented with a cell growth factor and a differentiation inhibitory factor to obtain EG cell colonies; (b) culturing the EG cells in the same medium as in step (a) by employing a feeder layer until the EG cells are colonized; and (c) recovering and subculturing the EG cells in the same medium as in step (a) to establish the EG cell line.

Description

field of invention [0001] The invention relates to a method for preparing an established avian pluripotent embryonic germ cell line and an avian pluripotent embryonic germ cell line. Background of the invention [0002] Embryonic stem (ES) cell lines are undifferentiated pluripotent cells isolated from blastocysts or morulae. Although ES cells are expected to be very useful, for example, for studies in developmental biology, to characterize totipotent cells, and for gene-targeting to generate genetically modified livestock, only mice with proven germline transmission have been established ES cells (Evans, M.J. and Kaufman, M.H., Nature, 292, 154-156 (1981); Bradley et al., Nature, 309, 255-256 (1984)), since germline Due to the limitations of transmission, the use of ES cells for livestock production has not been realized (First, N.L. et al., Reprod. Feril. Dev., 6, 553-562 (1994); Wheeler, M.B., Reprod. Fertil. Dev., 6, 563-568 (1994); Giles, J.R. et al., Mol. Reprod. Dev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N5/073C12N5/10C12Q1/02
CPCC12N5/0611C12N2501/105C12N2501/115C12N2501/125C12N2501/23C12N2510/00C12N2502/13
Inventor 韩在容朴泰燮
Owner 韩在容
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