Complete medium for cultivating cPGCs (chicken primordial germ cells) and using method of complete medium

Abstract

The invention discloses a complete medium for cultivating cPGCs (chicken primordial germ cells) and a using method of the complete medium. The complete medium consists of KO-DMEM, B-27supplement, NAA, nucleosides, GlutaMAX II, [beta]-mercaptoethanol, ovalbumin, OVT, heparin sodium, Activin A, TGF-[beta]1, BMP4, FGF-2 and IGF-1. The using method comprises the following steps: cultivating collected 14HH chicken embryo blood in a culture system containing a cPGCs complete culture solution, and conducting primary culture for 10-15d so as to obtain purified cells; (2) adding the purified cPGCs in the cPGCs complete medium once again and conducting in-vitro culture and amplification; and (3) conducting counting and indirect immunofluorescence detection on the cPGCs obtained from the in-vitro culture and amplification, including SSEA-1 and DAZL surface markers . The medium provided by the invention, different from a conventional culture method which has serum and a feed layer, is concrete in ingredients, and the medium can facilitate researches on a molecular mechanism of cell differentiation.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a complete culture medium for cultivating cPGCs and its application method. Background technique [0002] Primordial Germ Cells (PGCs) are the precursors of gametes and the centers of reproduction. Precise self-renewal of the germ cell lineage is fundamental to fertility and reproductive success. In avian species, PGCs form earlier during embryogenesis than in mammals, and it eventually develops into egg cells in females and spermatozoa in males. Migration pathway in embryos—transplanted into gonads after blood circulation, making it a target cell for cryopreservation and a good carrier for the preparation of transgenic poultry. Because PGCs can develop into functional gametes, it is of high research value as a carrier to carry foreign genes, make gonad chimeras and even transgenic animals. [0003] At present, a large number of chicken primordial germ cells (Chicken Primordial Ge...

AI Technical Summary

Problems solved by technology

Since the components of these secreted substances are not yet clear, there will be some interference in the study of the molecular mechanism of cell differentiation; in the study of gene modification, it is necessary to obtain cPGCs stably expressing the target gene through drug screening, usually using a feeder layer containing a resistance gene to carry out culture and screening, and this kind of culture with feeder layer system complicates the experiment, increases the difficulty of the operation of the experiment, and the damage to the feeder layer after drug screening will also affect the growth of cells; on the other hand, these cells need silk After treatment with mitomycin to block its mitosis, it can be used as feeder layer cells. Even a very small amount of mitomycin residue will affect the growth of cPGCs cells; Have a certain impact on cPGCs cells; animal-derived serum and feeder layer cell pollutants will contaminate the cells themselves

In addition, the use of feeder layers and the addition of serum also prolong the time of the experiment to a certain extent and increase the input of experiment funds

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