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Method for culturing chicken embryonic germ cells and special nutrient fluid thereof
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A germ cell and culture medium technology, applied to embryonic cells, germ cells, biochemical equipment and methods, etc.
Inactive Publication Date: 2009-05-06
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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Abstract
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Problems solved by technology
However, there are many differences between chicken embryonic development and mammalian embryonic development. Chicken eggs are located on the surface of the yolk. After fertilization, the male pronucleus is covered by the embryo. When the egg is laid, the embryo has developed into about 60,000 cells. blastoderm stage, which makes transgenic techniques that have been widely used in mammals, such as microinjection of fertilized eggs, not suitable for application in poultry
Method used
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[0101] 1. Take out 30 chicken eggs (Shouguang black chicken) that are due to hatch, make the air chamber end up, and disinfect the eggshells with iodine and alcohol.
[0102] 2. Use sterilized scissors to cut off the eggshell at the end of the air chamber, carefully remove the allantoic membrane, use a curved glass rod or tweezers with curved ends to reach under the neck of the embryo body, and gently stir up the chicken embryo and place it in a plate.
[0103] 3. Remove embryo eyes, blood clots, allantoic membrane, etc., and rinse chicken embryos with PBS.
[0104] 4. Place the chicken embryos in a plate filled with PBS, carefully remove the abdomen of the chicken embryos, remove the viscera in the abdomen, and expose the mesonephros and gonads. Pick out the gonad and its surrounding tissue with a glass needle under a stereomicroscope, and rinse with Hanks solution 2-3 times.
[0105] 5. Pour out the Hanks solution, and dige...
Embodiment 2
[0113] Embodiment 2, identification of EG cells
[0114] The EG cells cultured in Example 1 were passed to the fourth generation, and the following identifications were carried out:
[0115] 1. Morphological identification
[0116]EG cell clones grow in the shape of regular islands or colonies, distributed in multiple layers, and have obvious patterns. The cells are not closely arranged, and sometimes the internal cell boundaries can be seen clearly. EG-like cells contain large nuclei and relatively little cytoplasm, with inconspicuous nucleoli.
[0118] After EG cells were fixed in 1% glutaraldehyde for 5 min, washed twice with 1×PBS, immersed in periodic acidreagent at room temperature for about 5 min, then washed with 1×PBS, then immersed in Schiff’s reagent for about 15 min, 1×PBS After washing twice, the PAS-stained EG cells were observed under an inverted microscope.
[0119] EG cell colonies and individual PGCs were dark purple an...
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Abstract
The invention discloses a method for culturing chicken embryonic germ cells and a special culture solution thereof. The culture solution is a high sugar DMEM solution containing the following solutes: 3.5 to 4.5 g / L of glucose, 1.5 to 2.5 mM of L-glutamine, (4.5 to 5.5) x 10<5> M of beta-mercaptoethanol, 1 to 1.5 mM of hydroxyethyl piperazine ethyl sulfuric acid, 100 to 120 IU / ml of leukemia inhibitory factor, 4 to 6 ng / ml of stem cellgrowth factor, 10 to 12 ng / ml of basic fibroblast growth factor, 10 to 12 ng / ml of insulin-like growth factor-1, 10 to 15 percent of fetal bovine serum, 1 to 3 percent of chicken serum, 1 to 1.2 percent of chick embryo extract, and antibiotic. The method for culturing the chicken embryonic germ cells is to culture the chicken embryonic germ cells on a chicken embryonic fibroblast culture layer. Through the reformation and the modification of EG cells, new varieties of chicken can be effectively cultured.
Description
technical field [0001] The invention relates to a method for cultivating chicken embryo germ cells and a special culture solution thereof. Background technique [0002] There are two sources of embryonic stem cells with multidirectional differentiation potential: one is embryonic stem cells (embryonic stem cells, ES cells for short) from the inner cell mass of the blastocyst stage of early embryos, and the other is primordial germ cells from the embryonic gonads ( Primordial germ cells (PGCs) embryonic germ cells (embryonic germ cells, referred to as EG cells). Since Evans and Kaufman (1981) isolated ES cells from delayed implantation mouse early embryos, early embryos have been the only source for isolating clonal ES cells. Until 1992, Matsui et al. used the primordial germ cells of implanted mouse fetuses as materials to obtain EG cells, and for the first time more comprehensively confirmed that PGCs can also be used as raw materials for the isolation and clone of ES cell...
Claims
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IPC IPC(8): C12N5/06C12N5/0735
Inventor 杜立新李宏滨郝小静王娟
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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