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External Cultivating method for bird primordial germ cell

A technology of primordial germ cells and in vitro culture, applied in the direction of germ cells, biochemical equipment and methods, embryonic cells, etc., can solve problems such as subculture conditions that have not been explored

Inactive Publication Date: 2006-08-23
YANGZHOU UNIV
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

There are few research reports on the long-term culture and line establishment of chicken embryo PGCs, and the most suitable subculture conditions have not yet been explored.

Method used

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  • External Cultivating method for bird primordial germ cell
  • External Cultivating method for bird primordial germ cell
  • External Cultivating method for bird primordial germ cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0019] Step 1: Isolation of PGCs from chicken embryos

[0020] The fertilized eggs were hatched to the 19th stage, and the chicken embryos were aseptically obtained and soaked in PBS for 3 times. The head, tail, heart and hepatic processes were removed under a dissecting microscope, and the dorsolateral tissue of the anterior 1 / 3 of the hindgut (including the genital ridge) was removed, and the removed genital ridge and its surrounding tissues were soaked in PBS for 3 The second time, put it in a 75ml Erlenmeyer flask, add 5-10ml of 0.25% trypsin + 4% EDTA preheated at 37°C, digest at room temperature for 5min, and gently blow with a siliconized glass pipette to disperse. Digestion was terminated with 5% FCS in the digestion solution. Centrifuge at 200g for 8min, discard the supernatant, resuspend the cells with culture medium, perform trypan blue staining to calculate the number of viable cells, and adjust the cell concentration to 10 4 / ml was inoculated into culture flask...

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Abstract

The external cultivating method of primordial bird germ cell relates to transgene, animal cloning, rare animal preservation, tissue and cell engineering and other science fields. After it is inoculated to the CEF raising layer, the primordial bird germ cell is cultivated in liquid high-sugar DMEM cultivation medium with added calf serum in 10 úÑ, chicken serum in 2 úÑ, L-glutamine in 2mmol / L, sodium pyrovate in 1 mmol / L, beta ¿Cmercaptoethanol in 5.5í‡10-5 mol / L, optional amino acid 10 microliter / ml, hSCF in 15-20 ng / ml, mLIF in 10 U / ml, bFGF in 10-15 ng / ml, hIL-11 in 0.04 ng / ml, IGF in 10 ng / ml, and gentamycin sulfate in 100 U / ml. The present invention has simple operation and high repeatability, and lays the foundation of the relevant research.

Description

technical field [0001] The invention relates to the fields of transgene, animal cloning, preservation of rare animals, tissue and cell engineering and the like. technical background [0002] Bird transgenic technology has unique advantages and broad application prospects in improving bird production performance, breeding for disease resistance, and manufacturing bioreactors. Due to the unique reproductive physiology and anatomical characteristics of birds, the research on transgenic birds obviously lags behind that of mammals. For example, the microinjection method of mammals does not work in transgenic birds. Embryonic stem cells have pluripotency to form germline and somatic tissues, and can be genetically modified at the cellular level, so transgenic birds can be produced from embryonic stem cells. [0003] So far, there are two methods for culturing avian embryonic stem cells, one is to use blastodisc cell culture, and the other is to obtain from primordial germ cells (...

Claims

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Application Information

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IPC IPC(8): C12N5/06C12N5/0735
Inventor 李碧春秦洁孙鹏翔
Owner YANGZHOU UNIV
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