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Separation and purification method of Leopard Coral Grouper oogonium and identification and application thereof

A technology for separation and purification of oogonia, applied in cell dissociation methods, biochemical equipment and methods, germ cells, etc., can solve the problems of germplasm decline, limited male parental population, serious inbreeding, etc., and achieve cell viability High, universal equipment, small damage effect

Pending Publication Date: 2021-12-10
中国海洋大学三亚海洋研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Dongxingban is a hermaphroditic and proteogynous fish, which turns sexually male only at 4 to 5 years old, resulting in limited parental populations of existing male fish, severe inbreeding, and germplasm decline.

Method used

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  • Separation and purification method of Leopard Coral Grouper oogonium and identification and application thereof
  • Separation and purification method of Leopard Coral Grouper oogonium and identification and application thereof
  • Separation and purification method of Leopard Coral Grouper oogonium and identification and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0043] A method for isolating and purifying oogonia of Eastern star spot, comprising the following steps:

[0044] 1) Prepare a double-antibody DMEM medium with a penicillin concentration of 300IU / mL and a streptomycin concentration of 300μg / mL, and extract 4-month-old Eastern star spot ovaries into it. The mass ratio of double-antibody DMEM medium to ovary is 8:1 , placed at 4°C for 10 minutes, washed the ovary 3 times, and replaced with new DMEM medium;

[0045]2) Cut the ovary of step 1) from the side, remove the outer membrane of the ovary, cut the tissue into pieces, add 3 times the volume of DMEM medium, centrifuge at 50×g for 5 minutes, discard the supernatant, and add 3 times the volume of the precipitate to digest with protease The solution was digested at 25-30°C for 40-60 minutes, gently blown and mixed with a gun every 20 minutes, and the digestion situation was checked under a microscope, the ratio of digested cells and tissue blocks was counted, and the cell viab...

Embodiment 2

[0048] A method for isolating and purifying oogonia of Eastern star spot, comprising the following steps:

[0049] 1) Prepare a double-antibody DMEM medium with a penicillin concentration of 350IU / mL and a streptomycin concentration of 250μg / mL, and extract 3-month-old Eastern star spot ovaries into it. The mass ratio of double-antibody DMEM medium to ovary is 5:1 , placed at 4°C for 20 minutes, washed the ovary 3 times, and replaced with new DMEM medium;

[0050] 2) Cut the ovary from step 1) from the side, remove the outer membrane of the ovary, cut the tissue into pieces, add 3 times the volume of DMEM medium, centrifuge at 40×g for 8 minutes, discard the supernatant, and add 3 times the volume of the precipitate to digest with protease The solution was digested at 25-30°C for 40-60 minutes, gently blown and mixed with a gun every 10 minutes, and checked the digestion situation under a microscope, counted the proportion of digested cells and tissue blocks, and counted the c...

Embodiment 3

[0053] A method for isolating and purifying oogonia of Eastern star spot, comprising the following steps:

[0054] 1) Prepare a double-antibody DMEM medium with a penicillin concentration of 300IU / mL and a streptomycin concentration of 300μg / mL, and extract 6-month-old Eastern star spot ovaries into it. The mass ratio of double-antibody DMEM medium to ovary is 8:1 , placed at 4°C for 10 minutes, washed the ovary 3 times, and replaced with new DMEM medium;

[0055] 2) Cut the ovary of step 1) from the side, remove the outer membrane of the ovary, cut the tissue into pieces, add 3 times the volume of DMEM medium, centrifuge at 50×g for 5 minutes, discard the supernatant, and add 3 times the volume of the precipitate to digest with protease The solution was digested at 25-30°C for 40-60 minutes, gently blown and mixed with a gun every 20 minutes, and the digestion situation was checked under a microscope, the ratio of digested cells and tissue blocks was counted, and the cell via...

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Abstract

The invention provides a separation and purification method of a Leopard Coral Grouper oogonium and identification and application thereof. By adopting the separation and purification method, the oogonium with the proportion of 85.6-90.3% can be obtained from an ovary cell suspension with the oogonium proportion of about 10% through separation and purification; the method has small damage to the cell, the obtained cell is high in activity, the operation process is simple and convenient, used instruments are common, and the method is easy to popularize and use; according to the identification method of the Leopard Coral Grouper oogonium, a hybridization display technology is adopted, a Nanos2 probe is used for marking the oogonium, and the Leopard Coral Grouper oogonium can be quickly identified; the separation and purification method of the Leopard Coral Grouper oogonium provides a technical guarantee for Leopard Coral Grouper reproductive development research and later amplification of a Leopard Coral Grouper male individual by applying an oogonial stem cells with the help of a germ cell transplantation technology.

Description

technical field [0001] The invention relates to the technical field of seawater fish reproduction and cell culture, in particular to a method for separating and purifying oogonia and its identification and application. Background technique [0002] Fish oogonia are round or oval in shape, relatively large in size, with irregular nuclei and a large nuclear-to-cytoplasmic ratio, with 1 to 2 nucleoli. At present, only some fish oogonia have been analyzed and identified, and the identification, isolation and purification of grouper oogonia are rarely involved, and the research on grouper oogonia is also rarely reported. The research and application of grouper oogonia are limited. Therefore, the development of methods for the identification, isolation and purification of grouper oogonia is very important for the analysis and research of grouper oogonia, and it can also be used for the expansion of grouper oogonia by germ cell transplantation technology. Male fish individuals pr...

Claims

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Application Information

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IPC IPC(8): C12N5/075C12N5/02C12Q1/6834
CPCC12N5/0609C12Q1/6834C12N2509/00C12N2509/10C12Q2565/518C12Q2563/173Y02A40/81
Inventor 胡景杰包振民汪波吴绍轩丁晖王梦娅王铭翊
Owner 中国海洋大学三亚海洋研究院
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