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Method of establishing novel induction system to reprogram CEFs (chicken embryo fibroblasts) into PGCs (primordial germ cells)

A method of establishment and reprogramming technology, applied in biochemical equipment and methods, culture process, cells modified by introducing foreign genetic material, etc., can solve the problem that PGC is not enough to meet the needs of practical applications

Pending Publication Date: 2019-09-06
YANGZHOU UNIV
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Problems solved by technology

However, the PGC obtained from a single embryo is far from enough to meet the needs of practical applications
In particular, the source of PGC for endangered species has become a problem that limits this technology

Method used

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  • Method of establishing novel induction system to reprogram CEFs (chicken embryo fibroblasts) into PGCs (primordial germ cells)

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Embodiment Construction

[0025] Raw materials required in the present invention (brackets are manufacturers):

[0026] The OCT4, SOX2, NANOG, LIN28 overexpression vectors constructed by Jinkairui Company were packed with lentivirus, and the titer was 1×10 8 UI / mL; chick embryos hatched to 10 days old.

[0027] DMEM high glucose medium (Hyclone), FBS (Gibco), penicillin streptomycin (Beyantian), Polybrene (Beyantian), KO-DMEM (Gibco), chicken serum (Gibco), non-essential amino acids (sigma), β - Mercaptoethanol (sigma), hSCF (sigma), bfgf (sigma), Lif (sigma), BMP4 (Prospec), BMP8b (R&D Systems), EGF (R&D Systems), accutase (Gibco), SSEA-1 (R&D Systems) , Cvh (abcam), mitomycin C (sigma).

[0028] Required culture medium formula among the present invention is:

[0029] Culture solution A: DMEM high glucose medium, (volume concentration percentage) 5% FBS, (volume concentration percentage) 1% penicillin and streptomycin.

[0030] Culture medium B: Culture medium A, 5μg / ml polybrene (polybrene).

[...

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Abstract

The invention relates to a method of establishing a novel induction system to reprogram CEFs (chicken embryo fibroblasts) into PGCs (primordial germ cells) and belongs to the technical field of biology. In isolated primordial CEF, OCT4, SOX2, NANOG and LIN28 reprogramming factors are introduced to lentivirus exogenously; 21 days after continuous culture, iPSC (induced pluripotent stem cells) strain identification is performed with an SSEA-1 antibody; a positive cell line that is identified is replaced with a BMP4 / BMP8b / EGF induction system; iPGCs (induced primordial germ cells) are acquired byinduction. The problem is solved herein that the quantity of PGCs acquired in endangered species is low; germplasms resource protection and species restoration are achieved for the engendered chickenspecies.

Description

technical field [0001] The invention relates to a method for establishing a novel chicken CEF reprogramming induction system into PGC, and belongs to the field of biotechnology. Background technique [0002] In mammalian livestock, somatic cell nuclear transfer, in vitro fertilization and embryo transfer have all been successful, which can be combined with the cryopreservation of sperm, eggs, embryos and even somatic cells to achieve the protection of germplasm resources and species restoration. However, due to the characteristics of laying eggs, the above methods are difficult to realize on poultry. [0003] Primordial germ cells (PGCs) of poultry have the unique characteristic of settling in the gonads through blood migration, which makes the PGC migration path the only way to achieve the recovery of avian species. However, the PGC obtained from a single embryo is far from enough to meet the needs of practical applications. In particular, the source of PGC for endangered...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10
CPCC12N5/0611C12N2506/13C12N2501/603C12N2501/602C12N2501/605C12N2501/608C12N2533/52C12N2500/44C12N2501/125C12N2501/115C12N2501/235C12N2501/155C12N2501/11
Inventor 李碧春金晶左其生赵瑞丰李婷婷袁霞靳锴
Owner YANGZHOU UNIV
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