CN103789263B - Construction method of bastard halibut brain cell system - Google Patents
Construction method of bastard halibut brain cell system Download PDFInfo
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- CN103789263B CN103789263B CN201410071475.XA CN201410071475A CN103789263B CN 103789263 B CN103789263 B CN 103789263B CN 201410071475 A CN201410071475 A CN 201410071475A CN 103789263 B CN103789263 B CN 103789263B
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Abstract
The invention discloses a method for establishing a bastard halibut brain tissue-derived cell system. The method comprises brain tissue primary culture, subculture, cryopreservation, recovery and identification, wherein cell culture fluid adopted is obtained by adding fetal calf serum and human basic fibroblast growth factor to basal cell culture fluid. The established bastard halibut brain cell system is represented as an epithelioid-like astrocytic cell, which can support continuous passage to provide a great amount of bastard halibut brain cells and can be directly used for research of bastard halibut functional gene. The construction method disclosed by the invention not only is expected to serve as a platform for theoretical research of bastard halibut molecule cellular level but also can be used as an ideal material for research on fish hemadenology, reproductive biology and environmental internal-secretion interfering-substance toxicology.
Description
Technical field
The present invention relates to seawater fish cell culture technology, is a kind of construction process of lefteye flounder brain cell line specifically.
Background technology
Structure becomes already with cultivation animal cell line studies developmental biology, incretology, toxicology, genetics, virusology, immunologic powerful.So far, the fish cell system having nearly more than 280 strains different sets up in succession, plays a significant role in multiple fields such as physiology, virusology, toxicology, tumour and genetically engineereds.But comparatively speaking, the clone of seawater fish is also less, comprises saltwater fish and just only to have an appointment 100 strains.
Fish cerebral tissue is the vital tissue organ of fish, the regulation and control of its all vital movement is all limited by neural system and endocrine system, if the reproduction activities such as Gonad Differentiation, growth, maturation, gamete discharge are exactly lean on the endocrine feedback of cerebral tissue to regulate to control.Therefore, brain tissue cell is except having the using value of general clone in the research such as gene function analysis, virusology, the less fish ependymal lager functional study field probed into now can also be applied to, and Fish Endocrinology, reproductive biology, the toxicologic correlative study of environment incretion interferent.At present, fish brain cell line report is also less, and particularly fishes in bothid and true plaice there is not yet relevant report.
Lefteye flounder (Paralichthys olivaceus) is commonly called as tooth sheet fish, flatfish, flatfish, is under the jurisdiction of Osteichthyes, Actinopterygii, Pleuronectiformes, flounder suborder, lefteye flounder section, Paralichthys.It is Fresh & Tender in Texture, is a kind of famous and precious marine fish, is also one of the important marine culture and stock enhancement fish of Japan, Korea S and China.Therefore, the research of the aspect such as its developmental biology, reproductive biology, incretology, sex control becomes focus.Along with research is goed deep into, the effect of lefteye flounder colony-formation assays system in the problem in science such as cytobiology and gene function is resolved more and more draws attention.But, up to now, only there is lefteye flounder fin ray clone (Chinese Marine University both at home and abroad at present, virgin skirt is bright, Aquaculture, 1997,156:327-333.), embryo cell line (Huanghai Sea aquatic products institute of Chinese Fishery research institute, Chen Songlin, Diseases of Aquatic Organisms, 2004,60:241-246.), kidney cell system (Huanghai Sea aquatic products institute of Chinese Fishery research institute, Wang Na, Journalof Fish Diseases, 2011,34:81-85.) etc. 3 clones, there are no the foundation of this fingerling brain tissue cell system.Therefore, if externally lefteye flounder brain cell line can be set up, not only the effect in its sex determination, growth, breeding platform can be provided for researching fish ependymal lager function and nervus centralis endocrine system; And, material can be provided for the function of the pathogenesis of vitro study fish pathogens and immune disease-resistance gene; In-vitro screening experiment even also can be used for the endocrine disrupter toxicological study comparatively paid close attention at present, namely plays a significant role in the toxicologic study of ocean environment endocrine disrupter.Meanwhile, this method also goes for other fishes in bothid and true plaice other seawater fishs even.
Summary of the invention
The object of the present invention is to provide a kind of construction process of lefteye flounder brain cell line.
For achieving the above object, the present invention adopts technical scheme to be:
A kind of fish cerebral tissue derived cell system establishment method, comprises the following steps:
1) cell culture fluid is prepared: the MEM nutrient solution getting Hyclone company, the foetal calf serum accounting for cell culture fluid cumulative volume 20% is added in nutrient solution, 10ng/mL human alkaline fibroblast somatomedin, 100U/mL penicillin, 100 μ g/mL Streptomycin sulphates, pH value is 7.0-7.4, preserves at 4 DEG C, for subsequent use;
2) original cuiture: 0.5-1mL cerebral tissue fritter is joined in step 1) preparation cell culture fluid and suspend, then suspension is gone in culturing bottle and just puts cultivation in 25 ± 0.2 DEG C of incubators, until tissue block complete adherent after add the above-mentioned preparation cell culture fluid of 1-2mL again;
3) Secondary Culture: until above-mentioned original cuiture tissue block peripheral cell move out and breed to formed nido cell swoon time, adding 0.25% tryptic digestive juice makes cell hang, then utilize step 1) to prepare cell culture fluid and carry out former bottle Secondary Culture, after at the bottom of former bottle passage cell covers with bottle, go down to posterity with 1:1-1:2 sub-bottle, later 3-7 days goes down to posterity once; Reached for 10 generations, and then Establishment of Cell Line success.After reaching for 10 generations, the nutrient solution used that goes down to posterity is the step 1) preparation cell culture fluid that serum content is kept to cumulative volume 10%.At present, this cell reached for 40 generations.
Described fish are lefteye flounder, are also applicable to the structure as other fish brain cell lines such as turbot, verasper variegate simultaneously.
Described step 2) mesencephalic tissue fritter is about 1mm
3the fritter of size.
Described step 2) get the cerebral tissue of lefteye flounder under aseptic condition, after being placed in the Hyclone company MEM nutrient solution 3-5min adding penicillin, Streptomycin sulphate, sucking-off nutrient solution, repeatedly cerebral tissue is rinsed with 1-1.2mL PBS (pH7.2), sucking-off PBS, is cut into small pieces cerebral tissue again, stand-by.
Described step 3) until above-mentioned original cuiture tissue block peripheral cell move out and breed to formed nido cell swoon time, sucking-off nutrient solution, rinse with PBS (pH7.2), sucking-off PBS, add 0.25% trypsin solution digestion, then namely basis of microscopic observation is inhaled to cellular contraction and is abandoned trypsin solution, continue to examine under a microscope, treat cell rounding, in former bottle, add fresh step 1) preparation cell culture fluid make cell suspension, then suspension is gone down to posterity.
Advantage of the present invention and positively effect as follows:
1. the lefteye flounder brain cell line adopting the inventive method to build can carry out continuous passage, reached for 40 generations at present, a large amount of lefteye flounder brain cells can be provided, cell growth temperature is 25 ± 0.2 DEG C, cellular form is class Epithelial, growth curve is normal, and modal number is normal 48, can be applied to the research of the aspects such as fish cell biology, genetics, incretology, developmental biology, reproductive biology, environment incretion interferent toxicology and virusology; And, carry out gene transfection experiments with pEGFP-N3, stronger green fluorescence can be observed, confirm that lefteye flounder brain cell line can directly apply to foreign gene functional study.
2. the present invention is by easy handling, easy mode by the mode of cerebral tissue derived cell through original cuiture and Secondary Culture, obtains and continuous passage and cell can maintain the brain derived cell of good growth conditions.Specifically refer to that the construction process of the lefteye flounder brain cell line in the present invention is simple to operate, repeatability is strong, pancreatin in other fish brain cell lines foundation reported comparatively or collagenase digestion primary culture method are more easily grasped and operate, therefore this construction process be more suitable for build other fish particularly seawater fish cerebral tissue source clone, using value is very big.
Accompanying drawing explanation
Figure 1A, 1B are respectively the lefteye flounder brain cell line figure of Secondary Culture under the phase microscope that the embodiment of the present invention provides, and wherein A is lefteye flounder brain cell 3 generation figure (100 ×), and B is lefteye flounder brain cell 23 generation figure (100 ×).
The growth curve chart of the lefteye flounder brain cell line that Fig. 2 provides for the embodiment of the present invention.
Fig. 3 A, B, C are respectively the idiogram of the lefteye flounder brain cell line that the embodiment of the present invention provides, and wherein A is chromosome number distribution plan, and B is division phases (1000 ×), and C is caryogram.
Lefteye flounder brain cell immunofluorescence displaing micro picture (200 ×) that Fig. 4 provides for the embodiment of the present invention.
Fluorescent micrograph (100 ×) after the lefteye flounder brain cell line transfection pEGFP-N3 that Fig. 5 provides for the embodiment of the present invention.
Embodiment:
The present invention is optimized current fish cell system construction process, construction process is simple to operate, and repeatability is strong, and comparatively, the establishment method of other fish brain cell lines of report is more easily grasped and operates, using value is very big, for further experimental study provides material and technical support.
Below in conjunction with embodiment in detail structure of the present invention, qualification and methods for using them are described in detail.
Embodiment 1
The establishment method of lefteye flounder brain derived cell system, step is as follows:
1) cell culture fluid is prepared: get Hyclone company MEM nutrient solution, in nutrient solution, add the foetal calf serum accounting for cumulative volume 20%, 100U/mL penicillin, 100 μ g/mL Streptomycin sulphates, 10ng/mL rh-bFGF, pH value is 7.0-7.4, deposit for 4 DEG C, for subsequent use.
2) original cuiture: the aseptic cerebral tissue getting body weight 250g lefteye flounder, be placed in the addition of 400U/mL penicillin, 400 μ g/mL Streptomycin sulphates 4mL Hyclone company MEM nutrient solution sterile glass plate 3-5min after, sucking-off nutrient solution, cerebral tissue is rinsed twice with 1-1.2mL PBS (pH7.2), sucking-off PBS, is cut into about 1mm by cerebral tissue
3fritter, add 1mL above-mentioned steps 1) preparation cell culture fluid, cerebral tissue fritter is suspended, then goes to 25cm
2in culturing bottle, just put cultivation in 25 ± 0.2 DEG C of incubators.Within second day, in culturing bottle, supplement above-mentioned steps 1) preparation cell culture fluid 1mL, then changed in culturing bottle every 7 days and partly measures cell culture fluid once.
3) Secondary Culture: treat step 2) primary cultured cell constantly moves out and breeds rapidly around tissue block, when tissue block peripheral cell is dizzy stop growing after, sucking-off nutrient solution, rinses with PBS (pH7.2), sucking-off PBS, adds the 0.25% trypsin solution digestion of 1mL.Namely basis of microscopic observation is inhaled to cellular contraction and is abandoned trypsin solution, continues to examine under a microscope, treats cell rounding, adds fresh step 1) preparation cell culture fluid and makes cell suspension, then gone down to posterity by suspension in former bottle.After first time goes down to posterity, at the bottom for the treatment of that cell covers with bottle, with 2 × 10
4-2 × 10
5cell/mL concentration is carried out 1:1-1:2 sub-bottle and is gone down to posterity; Later 3-7 days goes down to posterity once; Reached for 10 generations, and then Establishment of Cell Line success.After reaching for 10 generations, the nutrient solution used that goes down to posterity is the step 1) preparation cell culture fluid that serum content is kept to cumulative volume 10%.At present, this cell reached for 40 generations.Cell can stablize increment, clone called after POBC.This clone major cell types is class epithelioid cell (as shown in Figure 1A, B).
Embodiment 2
The qualification of lefteye flounder brain cell line and application
1) the frozen and recovery of cell
Cell frozen:
Choose and be above-mentionedly in exponential phase of growth, cell density reach more than 90% Secondary Culture brain cell, digest according to a conventional method: sucking-off nutrient solution, rinse with PBS (pH7.2), sucking-off PBS, add the 0.25% trypsin solution digestion of 1mL.Namely basis of microscopic observation is inhaled to cellular contraction and is abandoned trypsin solution, continues to examine under a microscope, treats cell rounding, adds 2mL fresh above-described embodiment step 1) preparation cell culture fluid and prepare cell suspension in former bottle.Cell suspension is transferred in 15mL centrifuge tube, with 2200r
pthe centrifugal 2min of m, removes supernatant.With frozen storing liquid (namely containing the Hyclone company MEM cell culture fluid of the 10% dimethyl sulfoxide (DMSO)) suspension cell of 2mL precooling, and move in 2mL cryopreservation tube, 30min is placed in 4 DEG C successively after cryopreservation tube being put into program temperature reduction box, place 2h for-20 DEG C,-80 DEG C of placements are spent the night, then move in liquid nitrogen and preserve, and make a record.
The recovery of cell:
In liquid nitrogen, take out the cryopreservation tube of preservation, be placed in rapidly 42 DEG C of water-baths and melt, the centrifugal 5min of 2200rpm, abandons supernatant, and the above-mentioned freeze-stored cell and the cell culture fluid adding the preparation of 2mL above-described embodiment step 1) suspends, is transferred to 25cm
2in culturing bottle, be positioned over 25 ± 0.2 DEG C of cultivations in incubator, next day abandons supernatant after cell attachment, then adds the cell culture fluid of the new above-described embodiment step 1) preparation of 2mL.
2) cell growth curve is drawn
In order to analyze the growing state of lefteye flounder clone, get 22 generation brain cell, according to 1.2 × 10
5the density of individual cells/well inoculates 18 holes in 24 orifice plates, Tissue Culture Plate is placed in 25 DEG C of incubators and cultivates.Distinguish the 1st, 3,5,7,9 after inoculation, conveniently trypsin solution digestion method digestion in 11 days, collects 3 porocytes, counts with cell counting count board.Take incubation time as X-coordinate, with the cell quantity in every mL nutrient solution for ordinate zou, draw growth curve, as shown in Figure 2.
3) chromosome analysis
Get 25 generation lefteye flounder brain cell, suspend with the 2mL cell culture fluid prepared according to above-described embodiment step 1), be inoculated in 25cm
2culturing bottle in, inoculum density is 1 × 10
6individual cell/bottle.24h is cultivated at 25 ± 0.2 DEG C, after adding the colchicine 3h of final concentration 1 μ g/mL, conveniently trypsin solution digestion method digestion, the centrifugal 2min collecting cell of 2200rpm, after adding 10mL0.075mol/LKCl solution 37 DEG C of water-bath Hypotonic treatment 20min in cell precipitation, add the Ka Nuoshi stationary liquid (methyl alcohol: acetic acid=3:1(v/v) of 1mL precooling), pre-fix 2min, supernatant is abandoned after the centrifugal 5min of 1000rpm, after 15min fixed by cell precipitation 5mL precooling Ka Nuoshi stationary liquid, with the centrifugal 5min collecting cell of 1000rpm, repetitive operation twice.Then be resuspended in by cell in 0.5mL precooling Ka Nuoshi stationary liquid, cell suspension is dripped sheet with cold method, air-dry, 10% Giemsa staining 10min, uses Nikon microscopic examination.The chromosome number of result display lefteye flounder brain cell is between 29 ~ 93, and wherein modal number is 48 (Fig. 3 A), is telocentric chromosome, and karyotype is 2n=48t(Fig. 3 B, C).
4) cellular immunofluorescence qualification
Get 28 generation lefteye flounder brain cell, suspend, according to 1 × 10 with the 2mL cell culture fluid prepared according to above-described embodiment step 1)
5the density of individual cells/well is inoculated in 24 orifice plates, is placed in 25 ± 0.2 DEG C of incubators and cultivates.Treat that cell density reaches 95-100%, wash 3 times with precooling PBS (pH7.2), each 10min; Fix 10min with 4% paraformaldehyde, wash 5min with PBS; With the Triton perforation 15min of 0.5% precooling, wash 2 times with PBS, each 5min.Add primary antibodie (2.5 μ L Rabbit anti-GFAP [X] 18-0063 are dissolved in the 1%BSA of 200 μ L) 4 DEG C after closing 30min with 1%BSA after cleaning to spend the night; Wash 2 times with PBS, each 5min, then add two anti-(2.5 μ L Labeled Donkey AntiRabbit IgG Antibodies are dissolved in the 1%BSA of 200 μ L) 25 DEG C and hatch 1h, wash 2 times with PBS, each 5min, finally add 25 μ L DAPI and to dye 2min.With Nikon ECLIPSETE2000-U fluorescence microscope, find that there is the expression of green fluorescence, show that this cell is astroglia cell (Fig. 4).
5) expression of GFP reporter gene in brain cell
Day before transfection, get the 22nd generation brain cell, suspend with the 2mL cell culture fluid prepared according to above-described embodiment step 1), with 2 × 10
5the density of individual cells/well is inoculated in 24 orifice plates, 25 ± 0.2 DEG C of cultivations.During transfection, cell density more than 80%, with the Lipofectamine2000 transfection of Invitrogen company
peGFP-N3.Concrete steps are joined in the 1.5mL centrifuge tube of the MEM cell culture fluid comprising 49 μ L Hyclone companies by 1 μ L Lipofectamine2000, add 2 μ L pEGFP-N3(400ng/ μ L in another centrifuge tube) and the MEM cell culture fluid of 48 μ L Hyclone companies.After 5min, by above-mentioned two pipe solution mixing, room temperature places 25min.Above-mentioned 100 μ L mixed solutions are joined the cell culture fluid that comprises 500 μ L and do not prepare containing above-described embodiment step 1) of serum (namely not containing the preparation cell culture fluid of serum, get Hyclone company MEM nutrient solution, 100U/mL penicillin is added in nutrient solution, 100 μ g/mL Streptomycin sulphates, 10ng/mL rh-bFGF, pH value is 7.0-7.4, deposits for 4 DEG C, for subsequent use.) 24 orifice plates a hole in, after 25 ± 0.2 DEG C of cultivation 4.5h, sucking-off nutrient solution, add the cell culture fluid that 500 μ L prepare according to above-described embodiment step 1), after 72h, with the expression of Nikon ECLIPSE TE2000-U fluorescence microscope green fluorescence, find the cell expressing comparatively hyperfluorescenceZeng Yongminggaoyingguang (specifically see Fig. 5) about having more than 20%.
Above-described embodiment shows, the lefteye flounder cerebral tissue derived cell system set up by the inventive method, and growth curve is normal, and karyomit(e) is normal 48, can carry out continuous passage, also can carry out freezen protective to it.Carry out gene transfection experiments with pEGFP-N3, also observe stronger green fluorescence.Cellular immunofluorescence qualification result shows that institute's culturing cell is brain astroglia cell.Confirm that lefteye flounder cerebral tissue derived cell system can directly apply to foreign gene functional study.
The establishment method repeatability of lefteye flounder cerebral tissue derived cell system of the present invention is strong, and dyed body qualification result is credible, and the lefteye flounder body weight of drawing materials is appropriate, and working method is easy, also goes for the clone building other fish cerebral tissues source.
Claims (5)
1. an establishment method for lefteye flounder cerebral tissue astroglia cell system, comprises the following steps:
1) cell culture fluid is prepared: the MEM nutrient solution getting Hyclone company, the foetal calf serum accounting for cell culture fluid cumulative volume 20% is added in nutrient solution, 10ng/mL human alkaline fibroblast somatomedin, 100U/mL penicillin, 100 μ g/mL Streptomycin sulphates, pH value is 7.0-7.4, preserves at 4 DEG C, for subsequent use;
2) original cuiture: 0.5-1mL cerebral tissue fritter is joined step 1) preparation cell culture fluid in suspend, then suspension is gone in culturing bottle and just puts cultivation in 25 ± 0.2 DEG C of incubators, until tissue block complete adherent after add 1-2mL step 1 again) preparation cell culture fluid;
3) Secondary Culture: until above-mentioned original cuiture tissue block peripheral cell move out and breed to formed nido cell swoon time, adding 0.25% tryptic digestive juice makes cell hang, then utilize step 1) preparation cell culture fluid carry out former bottle Secondary Culture, after at the bottom of former bottle passage cell covers with bottle, go down to posterity with 1:1-1:2 sub-bottle, later 3-7 days goes down to posterity once; Reached for 10 generations, and then Establishment of Cell Line success.
2., according to the establishment method of lefteye flounder cerebral tissue astroglia cell system according to claim 1, it is characterized in that: described in reached for 10 generations after, the nutrient solution used that goes down to posterity is the step 1 that serum content is kept to cumulative volume 10%) preparation cell culture fluid.
3. according to the establishment method of lefteye flounder cerebral tissue astroglia cell system according to claim 1, it is characterized in that: described step 2) get the cerebral tissue of lefteye flounder under aseptic condition, after being placed in the Hyclone company MEM nutrient solution 3-5min adding penicillin, Streptomycin sulphate, sucking-off nutrient solution, cerebral tissue is rinsed more than 7.2 time with 1-1.2mL PBSpH, sucking-off PBS, is cut into small pieces cerebral tissue again, stand-by.
4., according to the establishment method of lefteye flounder cerebral tissue astroglia cell system according to claim 1, it is characterized in that: described step 2) mesencephalic tissue fritter is about 1mm
3the fritter of size.
5. according to the establishment method of lefteye flounder cerebral tissue astroglia cell system according to claim 1, it is characterized in that: described step 3) until above-mentioned original cuiture tissue block peripheral cell move out and breed to formed nido cell swoon time, sucking-off nutrient solution, rinse with PBS pH 7.2, sucking-off PBS, add 0.25% trypsin solution digestion, then namely basis of microscopic observation is inhaled to cellular contraction and is abandoned trypsin solution, continue to examine under a microscope, treat cell rounding, in former bottle, add step 1) preparation cell culture fluid make cell suspension, then suspension is gone down to posterity.
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| CN105200005A (en) * | 2014-08-13 | 2015-12-30 | 中国科学院海洋研究所 | Paralichthys olivaceus muscle satellite cell line establishing method, specific primer for identifying paralichthys olivaceus muscle satellite cell marker gene and application of specific primer |
| CN104630133B (en) * | 2015-02-05 | 2017-11-10 | 中国科学院海洋研究所 | A kind of construction method of lefteye flounder spermary cell system |
| CN105543162B (en) * | 2015-11-27 | 2020-03-31 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Crucian brain tissue cell line and application thereof |
| CN107686830B (en) * | 2017-10-19 | 2021-05-18 | 华中农业大学 | Method for culturing hypothalamic cells of grass carp in vitro |
| CN107699544A (en) * | 2017-10-19 | 2018-02-16 | 华中农业大学 | A kind of grass carp pituicyte extracorporeal culturing method |
| CN108753703B (en) * | 2018-06-11 | 2022-03-04 | 中国科学院海洋研究所 | Method for establishing paralichthys olivaceus embryonic muscle satellite cell line |
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| WO2012123117A1 (en) * | 2011-03-15 | 2012-09-20 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Process for producing fish cells with an elevated content of highly unsaturated fatty acids and use of these fish cells to obtain fish-specific products |
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