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Takifugu obscurus testis cell line and construction method and application thereof

A dark-patterned pufferfish, a construction method technology, applied in the direction of artificial cell constructs, applications, animal cells, etc., to achieve the effects of stable characteristics, strong proliferation ability, and good state

Pending Publication Date: 2022-07-29
HOHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the establishment of a stable cell line of puffer obscura at home and abroad. The testis cell line of puffer obscura is the first established cell line of puffer obscura, and it will become an important platform for in vitro research on gene function of puffer obscurus

Method used

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  • Takifugu obscurus testis cell line and construction method and application thereof
  • Takifugu obscurus testis cell line and construction method and application thereof
  • Takifugu obscurus testis cell line and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Isolation, culture and passage of testicular cells of pufferfish

[0046] (1) Cell isolation and primary culture: Take healthy pufferfish with a weight of 80-100 g, and soak the pufferfish in 75% alcohol for 3 min. After taking it out, it was placed in an ultra-clean bench, and the testis tissue was taken out under aseptic conditions, and rinsed three times in FBS containing 400 U / ml penicillin and 400 μg / ml streptomycin. Subsequently, the testis tissue was taken out and placed in a beaker, and the tissue was cut into small pieces with sterilized scissors, and 1 ml of 0.25% trypsin was added, and the tissue was cut into mince. After standing at room temperature for 15 min, the trypsin-digested cell suspension was filtered using a 40 μm cell sieve. The filtered cell suspension was centrifuged to remove the supernatant, and the cell pellet was collected. Cell culture medium, pipet into 25 cm 2 Cell culture flasks were cultured in a 28°C incubator. After 48 h ...

Embodiment 2

[0049] Example 2 Cryopreservation and recovery of testicular cell line of pufferfish with dark streaks

[0050] (1) Cryopreservation of cells: Select cells of different passages, when the cell growth density reaches 90%, according to the conventional method, aspirate the old medium, add 1ml of trypsin digestion solution to digest; when the cells are observed to shrink, aspirate the trypsin, Add 2 ml of fresh cell culture medium. The cells were then transferred to a 5 ml centrifuge tube, centrifuged at 1000 g for 5 min, and the supernatant was removed. Add 1 ml of cell cryopreservation solution, resuspend the cells and transfer into a cryopreservation tube. The cryovials were placed in a programmed cooling box, placed at -80 °C for 24 h, and then transferred to liquid nitrogen for long-term storage.

[0051] (2) Cell recovery: Take out the cells stored in liquid nitrogen, quickly place them in a 37°C water bath, and shake the cryopreservation tube constantly to thaw the cells...

Embodiment 3

[0052] Example 3 Determination of growth characteristics of testis cell line of pufferfish with dark stripes

[0053] (1) Determination of optimal serum concentration: cell culture medium containing fetal bovine serum FBS concentration of 5%, 10%, 15%, and 20% was prepared respectively. figure 2 The growth curves of the testis cell line of the pufferfish in the dark striae under different culture conditions: A is the medium with different serum concentrations; B is the different culture temperature conditions.

[0054] The 45th passage testicular cells were taken, and cell suspensions of four serum concentrations were prepared at a density of 2.5×104 cells / mL, inoculated in a 12-well plate, and cultured in an incubator at 28°C. At the same time every day thereafter, 3 wells of cells of each serum concentration were taken, digested by conventional trypsinization method, collected and counted with a cell counting plate, and counted continuously for 7 days. Taking the culture t...

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Abstract

The invention discloses a takifugu obscurus testis cell line as well as a construction method and application thereof, a physical shearing method is matched with a pancreatin digestion method to separate testis tissue cells, primary culture is carried out, in subculture, a nutrient-rich culture medium is used for subculture, and then the takifugu obscurus testis cell line is successfully constructed. The takifugu obscurus testis cell provided by the invention presents an epithelial cell form and is continuously cultured for more than 100 generations, a large number of takifugu obscurus testis cells can be provided, and the cells can maintain a good growth state and can be cryopreserved. The takifugu obscurus testis cell line provided by the invention can be directly used for regulating and controlling expression of genes related to sex differentiation and development of takifugu obscurus, and can also be used as an in-vitro research platform for functional genes of takifugu obscurus. The application value of establishing the testis cell line of the takifugu obscurus is remarkable, scientific research in the fields related to sex differentiation and development of the takifugu obscurus is facilitated, and development of the takifugu obscurus culture industry is facilitated.

Description

technical field [0001] The invention relates to a testis cell line of pufferfish with dark stripes, a construction method and application thereof, and belongs to the field of biotechnology. Background technique [0002] Takifugu obscurus, commonly known as puffer fish, belongs to Bonefish, Tetrodontiformes, Tetradontidae, and Takifugu, mainly distributed in the East China Sea, Yellow Sea, Bohai Sea and the middle and lower reaches of the Yangtze River. , is a migratory fish in seawater and freshwater, and can be cultured in freshwater environment. The dark-patterned puffer fish has delicious meat and is one of the famous "three fresh products of the Yangtze River" in my country. However, the ovary, liver, kidney and blood of the wild puffer fish are poisonous, while the testis, muscles and skin are non-toxic. At present, the resources of wild puffer fish are depleted day by day, and farmed puffer fish has become the main source. As a unique high-quality fishery germplasm r...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N15/85C12N5/10A01K61/10C12R1/91
CPCC12N5/0683C12N15/85A01K61/10C12N2509/10C12N2501/115C12N2501/11C12N2510/00Y02A40/81
Inventor 史燕高阳王超宇赵哲刘鑫梁夏颖
Owner HOHAI UNIV
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