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

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...

AI Technical Summary

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

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