CN114317419A - Method for constructing muscle cell line of Gymnocypris przewalskii - Google Patents
Method for constructing muscle cell line of Gymnocypris przewalskii Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The invention discloses a construction method of a muscle cell line of Gymnocypris przewalskii, which comprises primary culture, subculture, cell cryopreservation and recovery, wherein the cell culture solution of the primary culture comprises the following components: a basic culture medium, 10-30% of bovine serum, 200-400 IU/mL of penicillin, 200-400 mug/mL of streptomycin, 1-5 mmol/L of glutamine and 1-5 mmol/L of sodium pyruvate; the components of the subcultured cell culture solution comprise: a basic culture medium, 10-30% bovine serum, 50-250 IU/mL penicillin, 50-250 mug/mL streptomycin, 1-5 mmol/L glutamine and 1-5 mmol/L sodium pyruvate; the frozen stock solution for freezing the cells comprises the following components: a basic culture medium, 10-30% of bovine serum, 50-250 IU/mL of penicillin, 50-250 mug/mL of streptomycin, 5-30% of DMSO, 1-5 mmol/L of glutamine and 1-5 mmol/L of sodium pyruvate. The preparation method is simple, does not need special equipment, can be operated in a common sterile culture room, and the prepared cells have good and stable growth and can be used for researches on nutrition, immunity, environmental toxicity, gene function analysis and the like.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a construction method of a gymnocypris przewalskii muscle cell line.
Background
The bare carp (Gymnocyprizzewalski), commonly known as the flashfish, belongs to the order Cypriniformes, Cyprinidae, Schizothorax, and the genus Gymnocypris, and is mainly distributed in the Qinghai lake and the branch streams around the lake in China, is economic fish in low-temperature saline-alkaline waters of the plateau, is the only economic fish in the Qinghai lake, and is also one of important economic fishes in China. It is fond to inhabit in the beach side, the slow flowing water position between large stone piles, deep ponds or rock seams, has strong adaptability, and can live in brackish water (the salt content of the Qinghai lake water is 12-13 per thousand) or fresh water. Under natural conditions, the Gymnocypris przewalskii grows very slowly, and the average growth time of Gymnocypris przewalskii to 300-500 g is 7-10 years. From the 60 s of the 20 th century, hundreds of thousands of acres of grassland around the Qinghai lake are reclaimed into farmlands, 108 rivers flowing into the Qinghai lake are artificially blocked by rivers to block breeding channels, and a lot of rivers run dry and cut off, so that the Gymnocypris cannot lay eggs in fresh water, and a large amount of Gymnocypris die in estuary areas. The related data show that the quantity of the naked carp resources in the Qinghai lake is about 7500 tons, which is not enough to develop 1/10 at the initial stage, but at present, birds inhabiting the bird island need to swallow nearly thousands tons of naked carps every year, and in recent years, the quantity of the naked carps in the Qinghai lake is seriously reduced and the fishery resources are seriously damaged due to the pollution, damage, over-fishing and other reasons of the living environment of the naked carps, and meanwhile, the protection of the naked carps in the Qinghai lake faces important challenges along with the annual increase of the salinity of the Qinghai lake.
In order to protect Gymnocypris przewalskii resources, the Gymnocypris przewalskii artificial proliferation and release technology has been matured gradually in the last twenty years, the technology needs a Gymnocypris przewalskii parent matured under natural conditions as a sexual cell source for artificial insemination and hatching and breeding of larval fish, and the technology needs to be carried out in a specific time period. An artificial breeding technology for breeding Gymnocypris przewalskii is not established, and no report related to the total artificial breeding of Gymnocypris przewalskii groups exists at present, so that a large number of Gymnocypris przewalskii individuals are usually required when natural adaptation, evolution, diseases and other researches are carried out on Gymnocypris przewalskii, the source for obtaining Gymnocypris przewalskii individuals can only be carried out by capturing wild individuals, and the contradiction to urgent need to be solved is formed with the protection of germplasm resources of rare fishes.
The establishment of a gymnocypris przewalskii fish cell line as a cell model is an important method for solving the problems. Since the fish cell culture is started in the 60's of the 20 th century, more than 280 cell lines have been established so far, and the cell culture of Chinese fishes is over 30 years, only more than 50 cell lines have been established, and the species for establishing the cell lines is less than 1.5% of the total number of Chinese fishes (more than 3500 Chinese fishes), so that the establishment speed of the cell lines is very slow, and certain difficulties exist in the cell lines. Compared with other fish cell culture methods, the method has the advantages that the gymnocypris przewalskii individuals under natural conditions are generally diseased individuals and carry various parasites, so that pollution is more likely to occur in cell culture, the cell lethality rate is high, and the failure rate is increased. Therefore, a cell line construction method which has low cell lethality and can obtain a large amount of muscle cells and is used for researching basic research on the growth of Gymnocypris przewalskii is urgently needed.
Disclosure of Invention
The invention aims to provide a method for constructing a gymnocypris przewalskii muscle cell line, which can obtain a large number of muscle cells and is used for basic research on fish growth.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a construction method of a muscle cell line of Gymnocypris przewalskii, which comprises primary culture, subculture, cell cryopreservation and recovery, wherein the cell culture solution of the primary culture comprises the following components: a basic culture medium, 10-30% bovine serum, 200-400 IU/mL penicillin and 200-400 mug/mL streptomycin;
the components of the subcultured cell culture solution comprise: a basic culture medium, 10-30% bovine serum, 50-250 IU/mL penicillin and 50-250 mug/mL streptomycin;
the frozen stock solution for freezing the cells comprises the following components: a basic culture medium, 10-30% bovine serum, 50-250 IU/mL penicillin, 50-250 mug/mL streptomycin and 5-30% DMSO.
Further, the components of the cell culture solution of the primary culture include: a basic culture medium, 10-20% bovine serum, 200-300 IU/mL penicillin and 200-300 mug/mL streptomycin;
the components of the subcultured cell culture solution comprise: a basic culture medium, 10-20% bovine serum, 50-100 IU/mL penicillin and 50-100 mug/mL streptomycin;
the frozen stock solution for freezing the cells comprises the following components: a basic culture medium, 10-20% of bovine serum, 50-100 IU/mL of penicillin, 50-100 mu g/mL of streptomycin and 10-20% of DMSO;
still further, the components of the cell culture broth of the primary culture include: basic culture medium, 15% bovine serum, 300IU/mL penicillin and 300 mug/mL streptomycin;
the components of the subcultured cell culture solution comprise: basic culture medium, 15% bovine serum, 100IU/mL penicillin and 100 mug/mL streptomycin;
the frozen stock solution for freezing the cells comprises the following components: basal medium, 15% bovine serum, 100IU/mL penicillin, 100. mu.g/mL streptomycin, 10% DMSO.
The "%" is volume percentage concentration; the components in the culture medium are added according to the concentration, and the concentration unit is formed after each component.
Further, the basic culture medium in the primary culture and subculture is selected from one of DMEM (high glucose), DMEM (low glucose), L-15 and RPMI 1640 culture medium, and is preferably DMEM (high glucose) culture medium;
the basic culture medium in the frozen stock solution is DMEM (high glucose) culture medium;
the bovine serum is selected from one of fetal bovine serum and calf serum, and is preferably fetal bovine serum.
The minimal medium was purchased from Gibco company DMEM high-glucose medium with a product number of 11960044, containing 4500mg/L D-glucose, no L-glutamine, and no sodium pyruvate.
Furthermore, the components of the culture solution for primary culture, subculture and cell cryopreservation also comprise 1-5 mmol/L glutamine and 1-5 mmol/L sodium pyruvate.
Further, the components of the cell culture solution of the primary culture include: a basic culture medium, 10-20% of bovine serum, 200-300 IU/mL of penicillin, 200-300 mug/mL of streptomycin, 1-5 mmol/L of glutamine and 1-5 mmol/L of sodium pyruvate;
the components of the subcultured cell culture solution comprise: a basic culture medium, 10-20% of bovine serum, 50-100 IU/mL of penicillin, 50-100 mu g/mL of streptomycin, 1-5 mmol/L of glutamine and 1-5 mmol/L of sodium pyruvate;
the frozen stock solution for freezing the cells comprises the following components: a basic culture medium, 10-20% of bovine serum, 50-100 IU/mL of penicillin, 50-100 mug/mL of streptomycin, 10-20% of DMSO, 1-5 mmol/L of glutamine and 1-5 mmol/L of sodium pyruvate;
in a specific embodiment of the present invention, the components of the cell culture fluid of the primary culture include: basic culture medium, 15% bovine serum, 300IU/mL penicillin, 300 mu g/mL streptomycin, 2.5mmol/L glutamine, 2.5mmol/L sodium pyruvate;
the components of the subcultured cell culture solution comprise: basic culture medium, 15% bovine serum, 100IU/mL penicillin, 100 mug/mL streptomycin, 2.5mmol/L glutamine, 2.5mmol/L sodium pyruvate;
the frozen stock solution for freezing the cells comprises the following components: basic culture medium, 15% bovine serum, 100IU/mL penicillin, 100 mug/mL streptomycin, 10% DMSO, 2.5mmol/L glutamine, 2.5mmol/L sodium pyruvate.
Further, the step of primary culturing comprises: washing muscle tissue blocks with sterile PBS, inoculating to a culture bottle, performing inverted culture, adding 1ml of primary culture medium into the culture bottle to maintain the humidity in the culture bottle and prevent dry cells of the muscle tissue from dying, adding 3ml of cell culture solution the next day, and culturing to grow into a cell monolayer;
the primary culture conditions are as follows: and (3) inverting the mixture in an incubator at 22-26 ℃ for 24 hours, preferably inverting the mixture in an incubator at 25 ℃ overnight.
The muscle cell obtaining step comprises: and rinsing the larval fish of 3 months old by using sterile water, soaking the larval fish by using 75% alcohol for 40-100 s, and taking subcutaneous muscle tissues after drying.
Further, the step of subculturing comprises: and (3) spreading the primary muscle cells at the bottom of the bottle by 60%, sucking out primary culture solution, suspending the cells, adding cell culture solution, and performing subculture.
Further, the method for suspending cells is a trypsinization method; further, the reagent used in the trypsinization method comprises 0.2-0.4% of trypsin-EDTA, preferably 0.25% of trypsin-EDTA;
the subculture mode is as follows: the first passage is carried out according to the ratio of 1:1, then the passage is carried out according to the ratio of 1:2, the passage is carried out once every 8-12 days, and the passage is preferably carried out once every 10 days;
the temperature of the subculture is 22-26 ℃, and preferably 25 ℃.
Further, the step of cryopreserving the cells comprises: taking cells in logarithmic phase of growth, digesting with pancreatin, centrifuging, mixing the obtained precipitate with a frozen stock solution, resuspending, and preserving with liquid nitrogen;
the cell concentration after resuspension is 1X 105～1×107one/mL, preferably 1X 106one/mL.
Further, the step of resuscitating comprises: and (3) rapidly melting and centrifuging the frozen stock solution in water bath at 37 ℃, re-suspending the obtained precipitate by using a subculture cell culture solution, and culturing the precipitate at 25 ℃ until a cell monolayer grows.
The invention has the beneficial effects that:
(1) the muscle cells obtained by the method have low lethality, can obtain a large amount of muscle cells, and are convenient to be used for researches on nutrition, immunity, environmental toxicity, gene function analysis and the like of Gymnocypris.
(2) The method is simple and easy to implement, does not need special equipment, and can be operated in a common sterile culture room.
Drawings
FIG. 1 is a primary culture diagram;
FIG. 2 is a 20 th passage culture diagram.
Detailed Description
The technical solutions of the present invention are described clearly and completely below, and it is obvious that the described embodiments are some, not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without any inventive step, are within the scope of the present invention.
The DMEM high-sugar medium is purchased from Gibco company, has the product number of 11960044, and contains 4500mg/L D-glucose; fetal bovine serum was purchased from Shanghai Biotechnology, Inc., cat # E510008. The reagents used in the present invention are commercially available unless otherwise specified.
Example 1
(1) Muscle acquisition of Gymnocypris przewalskii
Selecting a gymnocypris przewalskii fry cultured in a pond for 3 months, soaking and rinsing the gymnocypris przewalskii fry for 3 times by using sterile water, soaking the fish body for 1 minute by using 75% alcohol, then airing, taking subcutaneous muscle tissue of the fish body, and placing the muscle tissue in sterile PBS for later use.
(2) Primary culture
The optimal basal medium is selected by performing experiments on 12 explants by adopting three basal media of DMEM (high glucose), Leibovitz' sL-15 and RPMI 1640.
The results indicate that cells can migrate from gill tissue in DMEM (high glucose) and Leibovitz's L-15 medium, exhibiting fibroblast or epithelial-like morphology. After culturing in DMEM (high-sugar) solution added with fetal bovine serum and penicillin streptomycin for 25 days, the monolayer cells removed from the explants can meet the requirement of passage for subculture. In contrast, in L-15 medium, the migration rate of the monolayer of cells around the tissue mass was too slow. Thus, DMEM (high sugar) was used as the basal medium during subsequent passages.
In order to obtain a large number of cells after 20 generations of subculture, the invention first prepares the following primary cell culture fluid: DMEM high-glucose medium (Gibco), 15% fetal bovine serum, 300IU/mL penicillin, 300. mu.g/mL streptomycin, 2.5mmol/L glutamine, 1.5mmol/L sodium pyruvate.
Cutting the obtained muscle tissue 1g into small pieces, each of which is 1mm2Rinsing with sterile PBS 3 times for 5 minutes each time,inoculating it to 25cm2Adding 1ml of primary culture medium into a cell culture bottle, inverting in an incubator at 23 ℃ overnight, adding 3ml of primary culture solution the next day, replacing the culture solution every three days, starting cell migration from a tissue block after 10 days, growing into a cell monolayer after 30-40 days, and covering the bottom of the culture bottle with 40-60% of the culture solution. The pollution can be controlled to be lower than 20% by the treatment method.
(3) Subculturing
Preparing a subculture cell culture solution according to the following formula: DMEM high-glucose medium (Gibco), 15% fetal bovine serum, 100IU/mL penicillin, 100. mu.g/mL streptomycin, 2.5mmol/L glutamine and 1.5mmol/L sodium pyruvate.
After 60% of muscle cells in primary culture are paved at the bottom of a culture bottle, subculture is started, and the steps are as follows: sucking out primary culture solution in a culture bottle, carrying out passage suspension on cells by using a trypsin digestion method of 0.25% trypsin-EDTA, adding 3mL of passage culture solution to neutralize trypsin for reaction, carrying out passage according to a ratio of 1:1 for the first time, carrying out passage according to a ratio of 1:2, continuously culturing in an incubator at 25 ℃, carrying out passage once every 10 days on average, and obtaining good and stable cells.
(4) Cell cryopreservation
Preparing a cell cryopreservation solution according to the following formula: DMEM high-glucose medium (Gibco), 15% fetal bovine serum, 100IU/mL penicillin, 100. mu.g/mL streptomycin, 10% DMSO, 2.5mmol/L glutamine and 1.5mmol/L sodium pyruvate.
After subculturing for 20 generations and successful establishment of muscle cell line (figure 2), taking cells in logarithmic growth phase, digesting with pancreatin, centrifuging cell suspension at 1200rpm for 5 minutes, discarding supernatant, adding prepared cell cryopreservation solution into cell sediment, resuspending, and controlling cell concentration to be 1 × 106～1×107And (4) transferring the 1m L cell suspension into a 1.8m L cryopreservation tube, placing the cryopreservation tube into a programmed cooling box, placing the tube in a liquid nitrogen atmosphere for long-term storage after placing the tube in a temperature of-80 ℃ for 24 hours.
(5) Cell resuscitation
Taking out the frozen cells from the liquid nitrogen, putting the frozen cells into a water bath kettle at 37 ℃, quickly shaking the frozen cells until the frozen cells are melted, and centrifuging the cell suspension at 1200rpm for 5 minutes to remove supernatant; and (4) resuspending the cells by using 1ml of subculture cell culture solution, transferring the cells into a cell culture flask, and culturing the cells in an incubator at 25 ℃ until the cells grow into a monolayer after 8-10 days.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A construction method of a muscle cell line of Gymnocypris przewalskii comprises primary culture, subculture, cell cryopreservation and recovery, and is characterized in that the components of a cell culture solution of the primary culture comprise: a basic culture medium, 10-30% of bovine serum, 200-400 IU/mL of penicillin and 200-400 mu g/mL of streptomycin;
the components of the subcultured cell culture solution comprise: a basic culture medium, 10-30% bovine serum, 50-250 IU/mL penicillin and 50-250 mug/mL streptomycin;
the frozen stock solution for freezing the cells comprises the following components: a basic culture medium, 10-30% bovine serum, 50-250 IU/mL penicillin, 50-250 mug/mL streptomycin and 5-30% DMSO.
2. The method of claim 1, wherein the components of the cell culture fluid of the primary culture comprise: a basic culture medium, 10-20% bovine serum, 200-300 IU/mL penicillin and 200-300 mug/mL streptomycin;
the components of the subcultured cell culture solution comprise: a basic culture medium, 10-20% bovine serum, 50-100 IU/mL penicillin and 50-100 mug/mL streptomycin;
the frozen stock solution for freezing the cells comprises the following components: a basic culture medium, 10-20% of bovine serum, 50-100 IU/mL of penicillin, 50-100 mu g/mL of streptomycin and 10-20% of DMSO;
further, the components of the cell culture solution of the primary culture include: basic culture medium, 15% bovine serum, 300IU/mL penicillin and 300 mug/mL streptomycin;
the components of the subcultured cell culture solution comprise: basic culture medium, 15% bovine serum, 100IU/mL penicillin and 100 mug/mL streptomycin;
the frozen stock solution for freezing the cells comprises the following components: basal medium, 15% bovine serum, 100IU/mL penicillin, 100. mu.g/mL streptomycin, 10% DMSO.
3. The method according to claim 1 or 2, wherein the basic medium in the primary culture and subculture is selected from one of dmem (high glucose), dmem (low glucose), L-15, RPMI 1640 medium, preferably dmem (high glucose) medium;
the basic culture medium in the frozen stock solution is DMEM (high glucose) culture medium;
the bovine serum is selected from one of fetal bovine serum and calf serum, and is preferably fetal bovine serum;
further, the DMEM (high glucose) medium was 4500mg/L D-glucose.
4. The construction method according to any one of claims 1 to 3, wherein the culture medium components of the primary culture, the subculture and the cell cryopreservation further comprise 1 to 5mmol/L glutamine and 1 to 5mmol/L sodium pyruvate.
5. The method of claim 4, wherein the components of the cell culture fluid of the primary culture comprise: a basic culture medium, 10-20% of bovine serum, 200-300 IU/mL of penicillin, 200-300 mug/mL of streptomycin, 1-5 mmol/L of glutamine and 1-5 mmol/L of sodium pyruvate;
the components of the subcultured cell culture solution comprise: a basic culture medium, 10-20% of bovine serum, 50-100 IU/mL of penicillin, 50-100 mu g/mL of streptomycin, 1-5 mmol/L of glutamine and 1-5 mmol/L of sodium pyruvate;
the frozen stock solution for freezing the cells comprises the following components: a basic culture medium, 10-20% of bovine serum, 50-100 IU/mL of penicillin, 50-100 mug/mL of streptomycin, 10-20% of DMSO, 1-5 mmol/L of glutamine and 1-5 mmol/L of sodium pyruvate;
further, the components of the cell culture solution of the primary culture include: basic culture medium, 15% bovine serum, 300IU/mL penicillin, 300 mu g/mL streptomycin, 2.5mmol/L glutamine, 2.5mmol/L sodium pyruvate;
the components of the subcultured cell culture solution comprise: basic culture medium, 15% bovine serum, 100IU/mL penicillin, 100 mug/mL streptomycin, 2.5mmol/L glutamine, 2.5mmol/L sodium pyruvate;
the frozen stock solution for freezing the cells comprises the following components: basic culture medium, 15% bovine serum, 100IU/mL penicillin, 100 mug/mL streptomycin, 10% DMSO, 2.5mmol/L glutamine, 2.5mmol/L sodium pyruvate.
6. The method of constructing according to claim 1, wherein the step of primary culture comprises: washing muscle tissue blocks with sterile PBS, inoculating to a culture bottle, performing inverted culture, adding 1ml of primary culture medium into the culture bottle to maintain the humidity in the culture bottle and prevent dry cells of the muscle tissue from dying, adding 3ml of cell culture solution the next day, and culturing to grow into a cell monolayer;
the primary culture conditions are as follows: and (3) inverting the mixture in an incubator at 22-26 ℃ for 24 hours, preferably inverting the mixture in an incubator at 25 ℃ overnight.
7. The method of construction according to claim 1, wherein the step of subculturing comprises: and (3) spreading the primary muscle cells at the bottom of the bottle by 60%, sucking out primary culture solution, suspending the cells, adding cell culture solution, and performing subculture.
8. The method of constructing according to claim 7, wherein the method of suspending cells is trypsinization; further, the reagent used in the trypsinization method comprises 0.2-0.4% of trypsin-EDTA, preferably 0.25% of trypsin-EDTA;
the subculture mode is as follows: the first passage is carried out according to the ratio of 1:1, then the passage is carried out according to the ratio of 1:2, the passage is carried out once every 8-12 days, and the passage is preferably carried out once every 10 days;
the temperature of the subculture is 22-26 ℃, and preferably 25 ℃.
9. The method of constructing according to claim 1, wherein the step of cryopreserving the cells comprises: taking cells in logarithmic phase of growth, digesting with pancreatin, centrifuging, mixing the obtained precipitate with a frozen stock solution, resuspending, and preserving with liquid nitrogen;
the cell concentration after resuspension is 1X 105～1×107one/mL, preferably 1X 106one/mL.
10. The construction method according to claim 1, wherein the resuscitation step comprises: and (3) rapidly melting and centrifuging the frozen stock solution in water bath at 37 ℃, re-suspending the obtained precipitate by using a subculture cell culture solution, and culturing the precipitate at 25 ℃ until a cell monolayer grows.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114410569A (en) * | 2021-03-11 | 2022-04-29 | 青海大学 | Construction method of gill cell line of Gymnocypris przewalskii |
| CN115595297A (en) * | 2022-09-22 | 2023-01-13 | 中国水产科学研究院南海水产研究所（Cn） | Trachinotus ovatus muscle cell line and construction method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757933A (en) * | 2012-08-07 | 2012-10-31 | 中国科学院昆明动物研究所 | Construction method of muscle cell line of anabarilius grahami |
| CN104805052A (en) * | 2015-04-27 | 2015-07-29 | 中国科学院昆明动物研究所 | Method for building neolissochilus banasi muscle cell line |
| CN104830760A (en) * | 2015-06-01 | 2015-08-12 | 中国海洋大学 | Turbot muscular cell line establishment method |
| CN108148803A (en) * | 2018-02-01 | 2018-06-12 | 中国水产科学研究院珠江水产研究所 | One plant of grass carp musculature cell line and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2020102488A4 (en) * | 2020-08-26 | 2020-11-19 | Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences | Gobiocypris rarus swim bladder cell line and culture method and induced transformation method thereof |
-
2022
- 2022-01-04 CN CN202210001963.8A patent/CN114317419B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757933A (en) * | 2012-08-07 | 2012-10-31 | 中国科学院昆明动物研究所 | Construction method of muscle cell line of anabarilius grahami |
| CN104805052A (en) * | 2015-04-27 | 2015-07-29 | 中国科学院昆明动物研究所 | Method for building neolissochilus banasi muscle cell line |
| CN104830760A (en) * | 2015-06-01 | 2015-08-12 | 中国海洋大学 | Turbot muscular cell line establishment method |
| CN108148803A (en) * | 2018-02-01 | 2018-06-12 | 中国水产科学研究院珠江水产研究所 | One plant of grass carp musculature cell line and application |
Non-Patent Citations (2)
| Title |
|---|
| WEI FULEI等: "Cell line derived from muscle of Gymnocypris przewalskii, a species of Schizothoracinae in Qinghai Lake, Qinghai–Tibet Plateau", IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY - ANIMAL, vol. 58, pages 970 * |
| 龙舒婷等: "鱼类细胞培养的方法、条件及应用研究概述", 生物学教学, vol. 45, no. 9, pages 4 - 5 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114410569A (en) * | 2021-03-11 | 2022-04-29 | 青海大学 | Construction method of gill cell line of Gymnocypris przewalskii |
| CN114410569B (en) * | 2021-03-11 | 2023-12-19 | 青海大学 | Construction method of Qinghai lake naked carp gill cell line |
| CN115595297A (en) * | 2022-09-22 | 2023-01-13 | 中国水产科学研究院南海水产研究所（Cn） | Trachinotus ovatus muscle cell line and construction method and application thereof |
| CN115595297B (en) * | 2022-09-22 | 2023-10-13 | 中国水产科学研究院南海水产研究所 | Trachinotus ovatus muscle cell line, construction method and application |
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