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Bastard halibut embryonic-period primordial germ cell tracking and positioning method

A technology of primordial germ cells and localization method, which is applied in the fields of biochemical equipment and methods, determination/inspection of microorganisms, etc., can solve the problems of cumbersome steps, unsuitable sampling and preservation methods, etc., to simplify the operation steps and facilitate the preservation of practical samples. method, signal detection effect is good

Inactive Publication Date: 2015-09-02
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

Whole-embryo in situ hybridization can track the migration and quantitative changes of PGCs during embryonic development, but this technology is mainly applied in freshwater fish that are easy to remove the egg membrane. The traditional method of sampling and preserving embryos before whole-body in situ hybridization Suitable for seawater fish embryo samples, and the steps are cumbersome

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  • Bastard halibut embryonic-period primordial germ cell tracking and positioning method

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Embodiment

[0027] 1. Sampling and washing. According to the timing of embryonic development, track the developmental status of fertilized eggs under a stereoscope, collect 0.5ml of samples at each critical stage, wash twice with RNase-free PBS buffer, and wash away the seawater, each time for 1-2min.

[0028] 2. Fixed. The washed embryo samples were placed in a 15ml RNAase-free centrifuge tube, and the volume ratio of the sample to 4% PFA was 1:10. After inversion and mixing, they were placed flat at 4°C and fixed for 24 hours.

[0029] 3. Long-term storage. Replace 4% PFA with 50% deionized formamide buffer (50% deionized formamide buffer is mixed with deionized formamide: RNase-free PBS buffer at a volume ratio of 1:1), place in Long-term storage at -20°C.

[0030] 4. Egg dissection. Under a stereoscopic microscope, add buffer solution containing 50% deionized formamide to the embryo sample to make the sample wet, and remove the egg membrane and yolk.

[0031] 5. Wash. After diss...

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Abstract

The invention relates to a positioning and marking method for embryonic-period primordial germ cells (PGCs), in particular to a bastard halibut embryonic-period primordial germ cell tracking and positioning method. The bastard halibut embryonic-period primordial germ cell tracking and positioning method includes the steps of fixing collected various periods of embryo samples of the bastard halibuts by a 4% PFA solution; using a PBS (phosphate buffer saline) solution with 50% of deionized formamide to preserve the embryo samples at the temperature of -20 DEG C, and subjecting the fixed and preserved embryo samples to oolemma removing, gradient methanol dewatering and rewatering; after rewatering, washing the various periods of embryo samples with PBS buffering liquid without RNA ( ribonucleic acid) enzyme, pre-hybridizing at the temperature of 62-65 DEG C for 2-4 hours; after hybridization, adding a hybridization solution with bastard halibut RNA probes into the various periods of embryo samples subjected to pre-hybridization for hybridizing overnight at the temperature of 62-65 DEG C; after hybridization, subjecting the various periods of embryo samples to washing, antibody incubation and rewashing, keeping away from light, and developing colors to achieve marking for tracking and positioning of the embryonic-period primordial germ cells of the bastard halibuts. The bastard halibut embryonic-period primordial germ cell tracking and positioning method has the advantages that the problems that yolks and oolemma of the samples hybridized in situ conventionally are difficult to strip and a background color is too deep after color developing detection are solved, and operation steps are simplified.

Description

technical field [0001] The invention relates to a method for positioning and marking embryonic PGCs, in particular to a method for early tracking and positioning of primordial germ cells of flounder. Background technique [0002] Flounder is a marine fish with high economic value in my country. Its meat is tender and nutritious, and it is very popular among consumers. Flounder has the characteristics that females grow faster than males and are larger in size. In recent years, with the breakthrough of gynogenetic technology in flounder, genetically female juvenile fish can be induced to obtain female or male-dominated populations by exogenous or environmental factor treatment during the critical developmental period, and flounder has gradually become a major player in the field of sexual differentiation research. Model species of economic fish. Whole-embryo in situ hybridization can track the migration and quantitative changes of PGCs during embryonic development, but this ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6841C12Q2543/10
Inventor 刘清华李军王学颖肖永双
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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