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Establishment and induced differentiation method of spermatogonial stem cell line of Chinese sword fish

A technology of stem cell differentiation and establishment method, which is applied in the field of establishment and induction of differentiation of equine spermatogonial stem cell lines, and can solve the problems of imperfect culture technology and the like

Active Publication Date: 2021-06-25
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In fish, due to the imperfection of SSCs in vitro culture technology, whether fish SSCs, like mammalian SSCs, have the characteristics of pluripotent stem cells that differentiate into different types of somatic cells remains to be further proved

Method used

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  • Establishment and induced differentiation method of spermatogonial stem cell line of Chinese sword fish
  • Establishment and induced differentiation method of spermatogonial stem cell line of Chinese sword fish
  • Establishment and induced differentiation method of spermatogonial stem cell line of Chinese sword fish

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Cultivation of Sperm Stem Cells of Snakefish

[0051] 1. Isolation and Digestion of Testis Cells from Snormouth Fish

[0052] Take juvenile horse mouth fish, soak the fish body in PBS containing 0.5% bleaching powder for 1 min, wash with PBS containing 1% double antibody, pH=7.4 for 3 times, and then dissect. Cut the belly of the fish with scissors, carefully clip out the testis with tweezers, place it in a petri dish containing 1% double-antibody PBS, carefully peel off the fat tissue on the surface of the testis with tweezers, wash 3 times with PBS containing 1% double-antibody, and transfer the testis Transfer to a 1.5mL EP tube containing 500μL of trypsin, cut the tissue properly with scissors, and place it on ice for digestion. After 1 h, 1 mL of medium was added to stop the digestion, and the tissue pieces were gently blown away with a pipette gun, and the cell suspension was transferred to a 24-well plate covered with 0.1% gelatin, and cultured at 28°C...

Embodiment 2

[0057] Example 2 Identification of Spermatogonial Stem Cells from Snakefish

[0058] 1. RT-PCR identification of testicular cell types in cultured Snormouth fish

[0059] In order to identify the cultured sago mouth testis cells as sago mouth spermatogonial stem cells, the RNA of the cells was extracted and reversed to cDNA for RT-PCR detection.

[0060] Primer Premier 6 was used to design primers for vasa, piwi, dnd, and dazl marker genes for spermatogonia, stem cell marker molecules nanog, gfrα1 primers, and dmrt primers for somatic cell marker genes (see Table 1 for primer sequence information). Cell RNA was extracted and reversed. Converted to cDNA, RT-PCR was used to identify the testicular cell types of the horse mouth fish. Take cells from four wells, trypsinize them, resuspend them in PBS into a 1.5mL EP tube, centrifuge at 3500g for 5min, wash once with PBS, carefully suck off the supernatant, and extract cell RNA according to the following steps. First, add 500 μL ...

Embodiment 3

[0082] Example 3 Inducing the Sperm Stem Cells to Differentiate into Somatic Cells

[0083] will be 5×10 5 Spermatogonia stem cells stably expressing red fluorescent protein were inoculated into a 24-well plate not coated with gelatin. Due to the poor adhesion ability of spermatogonia stem cells, the cells were suspended in the medium and gradually aggregated into clumps, forming pseudo Embryo bodies (such as Figure 6 B). Placed at 28°C for cultivation. Replace half of the medium every other day. After 7 days of suspension culture, add 5 mM retinoic acid to the medium to a final concentration of 10 μM, place the cells at 28°C for induction, replace half of the medium every other day, and continuously induce for 7 days. Transfer the formed spheroids to gelatin-coated cells. Adhesive culture was carried out in a 6-well plate, the spheroids were blown gently with a pipette gun to disperse the cells, and the differentiation of the spermatogonial stem cells of the horse mouth ...

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Abstract

The invention relates to the technical field of biology, in particular to establishment and induced differentiation methods of a spermatogonial stem cell line of opsariichthys bidens. The method comprises the following steps: (1) separating and digesting testis tissues of opsariichthys bidens, and performing planking culture on cells; (2) after the cells are adhered to the wall, carrying out passage, and continuously performing culturing for 45-60 generations. The spermatogonial stem cells of the opsariichthys bidens obtained by the method can be continuously passaged in vitro and can be differentiated into somatic cells of the opsariichthys bidens through induction, such as neuronal cells, epithelial cells, huge flat cells and astrocytes; and the cells can be induced and differentiated into sperms.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for establishing and inducing differentiation of a horse mouth fish spermatogonial stem cell line. Background technique [0002] Spermatogonial stem cells (SSCs) are the basis of spermatogenesis and are crucial to the transmission of genetic information. Fish, like other hermaphrodites, have a high capacity for self-renewal and differentiation in their SSCs, providing a stable source of germ cells for sperm production. In recent years, the research and application of fish SSCs in the fields of germplasm resource conservation, basic biology and germ cell transplantation have attracted extensive attention. However, due to the imperfection of SSCs culture technology, the SSCs of economic fish can only be cultured for a short period of time, and the stable passage of spermatogonial stem cell lines of economic fish has not been obtained, and the research on the differentiation p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/076C12N5/071C12N5/079C12N5/0793C12R1/91
CPCC12N5/061C12N5/0619C12N5/0625C12N5/0622C12N2509/00Y02A40/81
Inventor 李名友仲颖陈霄郭婧桂朗
Owner SHANGHAI OCEAN UNIV
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