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Spermatogonium separation medium and application thereof in separation and purification of oxyeleotris marmorata spermatogonium

A technology of Channa chinensis and spermatogonia, applied in the field of separation of fish germ cells, can solve the problems of high price, unsatisfactory separation and purification effect, etc.

Active Publication Date: 2021-04-06
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, using the existing methods to separate and purify the spermatogonial cells of Channa japonicus is not only expensive, but also unsatisfactory in the effect of separation and purification

Method used

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  • Spermatogonium separation medium and application thereof in separation and purification of oxyeleotris marmorata spermatogonium
  • Spermatogonium separation medium and application thereof in separation and purification of oxyeleotris marmorata spermatogonium
  • Spermatogonium separation medium and application thereof in separation and purification of oxyeleotris marmorata spermatogonium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 a kind of spermatogonia separation liquid

[0044] The spermatogonia separation solution includes testis digestion solution and percoll gradient solution.

[0045] The testis digestive solution includes the following components in mass concentration: L-15 medium is used as the base solution, 2mg / mL type IV collagenase, 2μg / mL DNase I, 0.25% trypsin, 0.02g / L EDTA, pH 7.2, 0.22 Sterilize with a μm membrane filter.

[0046] The percoll gradient solution is obtained by mixing the percoll dilution and 100% percoll stock solution in proportion;

[0047] Percoll gradient solutions include 15% percoll solution, 25% percoll solution and 40% percoll solution.

[0048] The percoll dilution involved in the above scheme is configured as follows:

[0049] 1) Phosphate buffered saline (PBS): 0.24g potassium dihydrogen phosphate, 1.44g disodium hydrogen phosphate, 8g sodium chloride, 0.2g potassium chloride, dissolved in 1L pure water, adjust the pH to 7.4, and filter t...

Embodiment 2

[0054] Embodiment 2 A kind of spermatogonia separation liquid

[0055] The spermatogonia separation solution includes testis digestion solution and percoll gradient solution.

[0056] The testis digestive solution includes the following components in mass concentration: L-15 medium is used as the base solution, 1 mg / mL type IV collagenase, 1 μg / mL DNase I, 0.20% trypsin, 0.02 g / L EDTA, pH 7.0, 0.22 Sterilize with a μm membrane filter.

[0057] Percoll gradient solution is obtained by mixing percoll dilution and 100% percoll solution in proportion;

[0058] Percoll gradient solutions include 8% percoll solution, 20% percoll solution, and 35% percoll solution.

Embodiment 3

[0059] Embodiment 3 a kind of spermatogonia separation liquid

[0060] The spermatogonia separation solution includes testis digestion solution and percoll gradient solution.

[0061] The testis digestive solution includes the following components in mass concentration: L-15 medium is used as the base solution, 3 mg / mL type IV collagenase, 3 μg / mL DNase I, 0.30% trypsin, 0.03 g / L EDTA, pH 7.5, 0.22 Sterilize with a μm membrane filter.

[0062] The percoll gradient solution is obtained by mixing the percoll dilution and 100% percoll solution in proportion:

[0063] Percoll gradient solution includes 18% percoll solution, 30% percoll solution, 45% percoll solution.

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Abstract

The invention belongs to the technical field of separation of fish germ cells, particularly relates to a spermatogonium separation medium and application of the spermatogonium separation medium in separation and purification of oxyeleotris marmorata spermatogonium, and provides the spermatogonium separation medium in order to develop a separation and purification method suitable for the oxyeleotris marmorata spermatogonium. The separation medium comprises testis digestive juice and a percoll gradient solution, wherein the testis digestive juice comprises an L-15 culture medium, IV-type collagenase, DNA enzyme I, pancreatin and EDTA, the percoll gradient solution is obtained by diluting 100% percoll storage liquid with a percoll diluent by different multiples, and the separation medium is used for separating and purifying the oxyeleotris marmorata spermatogonium. High-purity and high-activity oxyeleotris marmorata spermatogonium can be obtained and can be proliferated and differentiated in vitro.

Description

technical field [0001] The invention belongs to the technical field of separation of fish germ cells, and in particular relates to a spermatogonia separation liquid and its application in separating and purifying the spermatogonia of Channa japonica. Background technique [0002] Fish is an important biological resource. In recent years, due to habitat degradation and overfishing, fishery resources in natural waters have declined sharply, resulting in nearly 3,000 species of fish being endangered to varying degrees. Therefore, how to carry out long-term and effective protection of fish germplasm resources has become an urgent problem to be solved. [0003] Germ cells are the only cells that can transmit genetic material to the next generation, and are important germplasm resources and the basis for population reproduction. In the early stage of fish embryonic development, germ cells specialize to form primordial germ cells (primordialgerm cells, PGCs). Subsequently, PGCs ...

Claims

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Application Information

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IPC IPC(8): C12N5/076
CPCC12N5/061C12N2509/10
Inventor 刘威易梅生张洪张弯弯李文静莫承宇
Owner SUN YAT SEN UNIV
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