In vitro isolated culture method for rabbit melanophore

A technology for melanocyte separation and culture, which is applied in the field of in vitro separation and culture of rabbit melanocytes, can solve the problems of lack of cell research models in the genetic research of coat color, etc.

Inactive Publication Date: 2017-03-22
YANGZHOU UNIV
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Problems solved by technology

[0003] In order to solve the problem of the lack of suitable cell research models in the study of rabbit coat color genetics, the present invention provides a method for in vitro isolation and culture of rabbit melanocytes, which c

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  • In vitro isolated culture method for rabbit melanophore
  • In vitro isolated culture method for rabbit melanophore
  • In vitro isolated culture method for rabbit melanophore

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Embodiment 1

[0033] 1 Materials and methods

[0034] 1.1 Experimental materials

[0035] (1) Experimental animals: 6-month-old black rabbits.

[0036] (2) Test equipment: ophthalmic forceps, ophthalmic scissors, stainless steel filter (200 mesh), scalpel, etc.

[0037] (3) Main reagents: M254 / HMGS medium, PBS buffer, 0.25% trypsin digestion solution, 0.25% Dispase II digestion solution, SABC immunohistochemical staining kit, DAB chromogenic kit, Mouse Anti-S-100 , 0.1% TritonX-100, PBS buffer, fetal bovine serum (FBS), double antibody (penicillin and streptomycin mixture 100×), dimethyl sulfoxide (DMSO).

[0038] 1.2 Isolation and acquisition of rabbit melanocytes

[0039] (1) Rabbits were anesthetized with barbiturate sodium solution, the sampling area was disinfected with 75% alcohol, the back hair was shaved off with shears, a piece of skin tissue of 1.5 cm × 1.5 cm was taken, and washed three times with pre-cooled PBS containing double antibodies.

[0040] (2) Put the skin tissue i...

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Abstract

The invention relates to an isolated culture method for primary generation rabbit melanophore. According to the method, the rabbit melanophore is isolated through an enzyme digestion method, and after culturing is conducted through an M254 complete medium, an M254/HMGS culture medium is adopted through replacement for culturing till the convergence degree reaches 80%-90%, then a differential digestion method is utilized for removing other cells, and the purified melanophore is obtained. The purity of the melanophore is greatly improved, the cultured cells are identified through cell morphologic observation, L-DOPA staining, semiquantitation and an anti-S-100 immunocytochemical staining method, the result shows that the isolated primary generation melanophore is mostly in a dendritic shape and a thin and long shuttle shape, the brownish black is shown after L-DOPA staining, the RT-PCR analysis shows that the isolated cells express the marker gene of the melanophore, and the S-100 staining cytoplasm is brown.

Description

technical field [0001] The invention relates to the fields of cytogenetics and molecular biology, in particular to a method for separating and culturing rabbit melanocytes in vitro. Background technique [0002] Mammalian skin and coat have a variety of colors, which are caused by chemically stable melanin, which can be divided into pheomelanin and eumelanin, which are oxidized by tyrosine or related components under the catalysis of corresponding enzymes. into. Inherited genes control the formation and type of coat color pigments that determine the coat color of mammals. As an important fur animal and experimental model, rabbits are also very rich in fur colors, including black, gray, yellow, blue and other natural colors, without printing and dyeing, and are favored by consumers. However, due to the difficulties in live animal experiments and the lack of suitable cell models, it has seriously hindered people's research on the genetic mechanism of rabbit coat color. This...

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0626C12N2509/00
Inventor 陈阳穆琳赵博昊胡帅帅赵斌杨乃苏王曼曼郝晔朱杰吴信生
Owner YANGZHOU UNIV
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