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Positioning marking method for primordial germ cells (PGCs) of scophthalmus maximus at embryonic development stage

A technology of primordial germ cells and positioning markers, applied in the field of PGCs positioning markers, can solve the problems of limiting the development of PGCs cryopreservation technology, unclear location and quantity of PGCs, inapplicable sampling and storage methods, etc., to achieve convenient collection and preservation process, Improve the permeability and facilitate the effect of going anywhere

Active Publication Date: 2015-01-14
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

However, the position and quantity of PGCs in seawater fish during embryonic development are still unclear, which greatly limits the development of PGCs cryopreservation technology.
Whole-embryo in situ hybridization can track the migration and quantity changes of PGCs during embryonic development, but this technology is mainly used in freshwater fish that are easy to remove the egg membrane. Suitable for seawater fish embryo samples, and the steps are cumbersome

Method used

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  • Positioning marking method for primordial germ cells (PGCs) of scophthalmus maximus at embryonic development stage
  • Positioning marking method for primordial germ cells (PGCs) of scophthalmus maximus at embryonic development stage

Examples

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Embodiment 1

[0026] 1) Collect 1ml of embryo samples at each stage and place them in 15ml centrifuge tubes, then wash them twice with 1×PBST (DEPC treated) buffer, 5min each time;

[0027] 2) After washing, fix with 13ml of 1×PBST (DEPC treated) solution containing 4% (mass ratio) paraformaldehyde at 4°C for 24h;

[0028] 3) Then use 10ml of 1×PBST (DEPC treated) solution containing 50% (volume ratio) formamide to store at -20°C (long-term storage is possible).

[0029] 4) After fixation, take 30 embryos at different developmental stages and place them in 1×PBST (treated with DEPC) under a dissecting microscope to remove the membrane and yolk with tweezers;

[0030] 5) Take the embryos at different developmental stages without membranes and yolk into 1.5ml centrifuge tubes, wash with 1×PBST (DEPC treatment) for 3 times, each time for 5min; 1*PBST, 1×PBST containing 50% (volume ratio) methanol, 1×PBST containing 75% (volume ratio) methanol, each treated with gradient methanol for 5 minutes...

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Abstract

The invention relates to a positioning marking method of PGCs, and concretely relates to a positioning marking method for primordial germ cells (PGCs) of scophthalmus maximus at embryonic development stage. The method comprises: acquiring embryo samples of scophthalmus maximus at all stages, fixing, using methanamide with the concentration of 50% to store the samples at -20 DEG C to 40 DEG C for usage; removing oolemma of all above embryo samples of scophthalmus maximus at all stages, using 1*PBST-methanol eluants with different concentrations to perform gradient dewatering processing, and storing at -20 DEG C to -40 DEG C; employing gradient methanol o process the embryo samples at all stages and subjected to above dewatering processing for rewatering, then using a 1*PBST buffer to wash, and after eluting, pre-hybridizing at 60-65 DEG C for 1-2 h; after prehybridization, hybridizing in a hybridization liquid containing a scophthalmus maximus RNA probe at 60-65 DEG C for a night; and after hybridization, washing, performing antibody incubation and color development, and further performing positioning marking. The invention provides the relatively convenient and practical sample storage method, the problem that oolemma and yolk are difficult to remove because methanol dewatering and storage are performed during conventional embryo integral in-situ hybridization is solved, and a protease K digestion step is saved, and the operation steps are simplified.

Description

technical field [0001] The invention relates to a method for positioning and marking PGCs, in particular to a method for positioning and marking primordial germ cells (PGCs) in the developmental stage of turbot embryos. Background technique [0002] Turbot is one of the most important marine aquaculture economic species in northern my country. However, due to the rapid development of the industry in the past ten years, germplasm resources have degraded, diseases have increased, and aquaculture benefits have declined. Therefore, it is necessary to protect its germplasm resources. Although sperm cryopreservation technology is relatively mature, there has been no breakthrough in research on cryopreservation of fish embryos, which is also a difficult problem in the preservation of international germplasm resources. The cryopreservation of PGCs provides a new breakthrough for the protection of germplasm resources. However, the location and quantity of marine fish PGCs during em...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/02
CPCC12Q1/6841C12Q2543/10
Inventor 刘清华李军林帆徐世宏
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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