Trachemys scripta elegans embryo fibroblast cell line and construction method thereof

A technology of fibroblasts and construction methods, applied in the field of red-eared turtle embryonic fibroblast cell lines and their construction, can solve the problems of immaturity and lack of in vitro culture technology of red-eared turtle cells, achieve low differentiation, fast growth, The effect of high passage times in vitro

Active Publication Date: 2018-03-30
INST OF ZOOLOGY GUANGDONG ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Studies have shown that red-eared turtles have strong tolerance to temperature changes, starvation, high-salt stress and acid stress. The reason may be tha

Method used

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  • Trachemys scripta elegans embryo fibroblast cell line and construction method thereof
  • Trachemys scripta elegans embryo fibroblast cell line and construction method thereof

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Effect test

Embodiment 1

[0021] 1 Materials and methods

[0022] 1.1 Collection of red-eared turtle embryos

[0023] The fertilized eggs of the red-eared turtle were collected from the turtle farm in Foshan, Guangdong. The collected turtle eggs were buried in vermiculite (the mass ratio of vermiculite to water: 1:0.8), and transferred to the laboratory within 24 hours after laying eggs. Incubation in a wet incubator at a temperature of 32°C and a humidity of 75% to 85%. After 5 to 6 days of hatching, the turtle eggs without obvious fertilization spots were discarded, and the other fertilized eggs continued to hatch.

[0024] 1.2 Biochemical reagents

[0025] Fetal bovine serum, 0.25% trypsin (Trypsin), DMEM, Opti-MEM (purchased from Gibco); DMSO, 100×penicillin-streptomycin solution (penicillin 10kU / mL, streptomycin 10mg / mL), PBS, MTT Cell Proliferation Detection Kit (purchased from Sangon Bioengineering Co., Ltd.); Lipofectamine 3000 (purchased from Thermo Company).

[0026] 1.3 Experimental meth...

Embodiment 2

[0044] 1 Materials and methods

[0045] 1.1 Collection of red-eared turtle embryos

[0046] The fertilized eggs of the red-eared turtle were collected from the turtle farm in Foshan, Guangdong. The collected turtle eggs were buried in vermiculite (the mass ratio of vermiculite to water is 1:0.8), and transferred to the laboratory within 24 hours after laying eggs with constant temperature and humidity. Incubation in an incubator at a temperature of 32°C and a humidity of 75% to 85%. After 5 to 6 days of hatching, the turtle eggs without obvious fertilization spots were discarded, and the other fertilized eggs continued to hatch.

[0047] 1.2 Biochemical reagents

[0048] Fetal bovine serum, 0.25% trypsin (Trypsin), DMEM, Opti-MEM (purchased from Gibco); DMSO, 100×penicillin-streptomycin solution (penicillin 10kU / mL, streptomycin 10mg / mL), PBS, MTT Cell Proliferation Detection Kit (purchased from Sangon Bioengineering Co., Ltd.); Lipofectamine 3000 (purchased from Thermo Com...

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Abstract

The invention discloses a trachemys scripta elegans embryo fibroblast cell line and a construction method thereof. The construction method comprises the following steps: taking a trachemys scripta elegans egg hatched for 12 to 18 days, selecting a live embryo and then sterilizing the trachemys scripta egg; shelling, taking the trachemys scripta embryo, discarding the head, fours limbs and visceraof the trachemys scripta embryo, and flushing the rest embryonic tissues with a PBS (Phosphate Buffered Saline) containing double-antibody until no obvious bloodstain is found; using trypsin to digestthe embryonic tissues which are flushed until no obvious bloodstain is found, collecting digested monoplast, and inoculating the monoplast into a complete medium for static culture; when the cells are partially adherent and do not float by slightly oscillating, absorbing the complete medium and the cells which are not adhered, and adding the rest adherent cells into a new complete medium for continuously culturing; when the confluence of primary cultured cells reaches 90 percent or above, digesting with the trypsin, collecting the monoplast and then inoculating into the complete medium for subculture. The invention lays a material and technical foundation for further using RSTEFs for conducting basic theory and experimental research.

Description

Technical field: [0001] The invention belongs to the technical field of cell culture, and in particular relates to a red-eared turtle embryonic fibroblast cell line and a construction method thereof. Background technique: [0002] The red-eared turtle (Trachemys scripta elegans), also known as the Brazilian turtle, is native to the central part of the United States to the northern part of Mexico. Due to international trade, this species has been introduced to many countries such as Europe, Africa, Australia, Asia and the Americas, and has become a worldwide species. Dangerous invasive species. At the same time, red-eared turtles are cheap and abundant, and they are also experimental animals for zoology, physiology, and medical research. Studies have shown that red-eared turtles have strong tolerance to temperature changes, starvation, high-salt stress and acid stress. The reason may be that the in vitro culture technology of red-eared turtle cells is immature, and there is...

Claims

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Application Information

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IPC IPC(8): C12N5/073C12N15/85C12N15/65
CPCC12N5/0603C12N5/0656C12N15/65C12N15/85C12N2800/106
Inventor 曹代男段好冉魏玉峰杨江波万小娟葛研龚世平
Owner INST OF ZOOLOGY GUANGDONG ACAD OF SCI
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