CN104004707A - Pseudosciaena crocea muscle cell line and its establishing method - Google Patents
Pseudosciaena crocea muscle cell line and its establishing method Download PDFInfo
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Abstract
The invention discloses a Pseudosciaena crocea muscle cell line and its establishing method. The cell line is preserved in China Center for Type Culture Collection on Oct., 31, 2013, and has a preservation registration number of CCTCC NO:C2013158. The Pseudosciaena crocea muscle cell line can be serially passed, can provide a large amount of the Pseudosciaena crocea muscle cell line, and can be used for researching pathogenic characteristics, vaccines, gene functions and breeding; and the Pseudosciaena crocea muscle cell line has excellent properties, is a fibroblast and is soma-fusiform or anomaly triangular, the cytoplasm outwardly stretches to form a plurality of protrusions with different lengths, and the protrusions are arranged in a radial or spiral manner. The Pseudosciaena crocea muscle cell line has the characteristics of strong splitting, short passage time, easy digestion, good adherent rate and starvation resistance. The establishing method has strong repeatability, and the established cell line has a good stability.
Description
Technical field
The invention belongs to fish cell culture technique field, be specifically related to a kind of muscles of Pseudosciaena crocea clone and establishment method thereof.
Background technology
Large yellow croaker (Larimichthys crocea), Osteichthyes, Perciformes, Sciaenidae, yellow croaker belongs to.Fresh & Tender in Texture, economic worth is higher, and market demand is larger.Before the seventies in last century, wild large yellow croaker resource is more abundant, and annual amount of fishing was once reaching the level of 120,000 tons.Due to wild large yellow croaker is especially dragged for excessively capturing disorderly of its Wintering group, cause wild large yellow croaker output exhausted rapidly the beginning of the seventies.After the eighties, along with reasons such as coastal waters economic development, environmental pollutions, cause the no longer suitable large yellow croaker in former each large spawning ground in coastal waters to lay eggs, existing wild large yellow croaker quantity of the catch is extremely low.Within 1986, large yellow croaker artificial breeding is broken through, and obtains subsequently paying attention to fast development, present stage market large yellow croaker be all to be provided by sea farming large yellow croaker
[84].Yet present stage is followed industrialization development, coastal waters breeding environment further worsens; Large yellow croaker disease takes place frequently; Large yellow croaker parent population resource exhaustion, the factors such as germplasm degeneration have limited further developing of large yellow croaker cultivation greatly, present stage approximately 8.6 ten thousand tons of large yellow croaker cultured outputs, not as good as the amount of fishing before the seventies.Therefore very urgent to the research of large yellow croaker disease control, fine-variety breeding, genetic background.
Setting up fish cell system can be for the researchs such as fishes virus separation, Antiviral Mechanism, vaccine research and development, transgenic technology, mosaic organizational project provide convenient greatly, and the relative experiment made on the living of resource requirement such as facility manpower is low, reproducible, and research cycle is short.Foundation about large yellow croaker clone at present only has grandson to like to have set up fin, kiss end, three kinds of clones of spleen of large yellow croaker adult fish at report in 2010, and has no the report of other tissue lines.
Summary of the invention
The object of the present invention is to provide a kind of muscles of Pseudosciaena crocea clone.
Another object of the present invention is to provide the establishment method of above-mentioned muscles of Pseudosciaena crocea clone.
Technical scheme of the present invention is as follows:
A kind of muscles of Pseudosciaena crocea clone (being the muscles of Pseudosciaena crocea clone LYCMS in biomaterial preservation proof), this cell lie in the Chinese Typical Representative culture collection center that is deposited on October 31st, 2013 (China. Wuhan. Wuhan University), preservation is registered on the books and is numbered: CCTCC NO:C2013158.
Another technical scheme of the present invention is as follows:
An establishment method for above-mentioned muscles of Pseudosciaena crocea clone, comprises the steps:
(1) obtaining of muscles of Pseudosciaena crocea tissue: large by 7 months, (penicillin and Streptomycin sulphate are dual anti-purchased from Corning Incorporated containing penicillin and the dual anti-sterilizing seawater of Streptomycin sulphate for the Pseudosciaena crocea Fry of long 9-11cm, this dual anti-100X concentrated solution is containing 10000U/mL penicillin and 10000 μ g/mL Streptomycin sulphates, preparation during seawater every 100mL seawater add 1mL penicillin and the dual anti-100X concentrated solution of Streptomycin sulphate) in support temporarily 1-1.5 hour, then splash into the Eugenol that accounts for sterilizing seawater volume 0.005-0.015%, until fish body upset, belly upward, to stimulate stress not behavior till; With sterile gauze piece, wipe fish surface mucus, to soak behind spirituous cotton balls wiping fish surface, take out muscles of Pseudosciaena crocea tissue and be placed in containing penicillin and the dual anti-D-hanks liquid (100mL D-hanks liquid adds 1mL penicillin and the dual anti-100X concentrated solution of Streptomycin sulphate) of Streptomycin sulphate;
(2) former culture: above-mentioned muscles of Pseudosciaena crocea tissue is shredded to 0.7-2.0mm
3small tissue blocks, with the rinsing of D-hanks liquid, finally with complete culture solution rinsing; After rinsing, above-mentioned small tissue blocks is inoculated in Tissue Culture Flask, absorb unnecessary nutrient solution, 27 ℃ of constant temperature dry doublings of upset body 24 hours, add again complete culture solution, right body so that small tissue blocks immerses in nutrient solution, in 27 ℃ of constant temperature culture, start former culture, change weekly complete culture solution once;
(3) cultivation of going down to posterity: primary while being cultured to attached cell propagation to 50-60% fraction of coverage at least, remove old nutrient solution, and clean and remove residual serum and divalent-metal ion; Then with 0.25% trypsin digestion, go down to posterity and hanged cell, with 1:2-10, go down to posterity; Every 2-8 days, go down to posterity once later, described muscles of Pseudosciaena crocea Establishment of Cell Line success.
In a preferred embodiment of the invention, described complete culture solution comprises: DMEM/F12 nutrient solution, foetal calf serum FBS, beta-mercaptoethanol, N-acetyl glucose osamine, Xylo-Mucine, people FGF-basic, people EGF, people HGF, penicillin and Streptomycin sulphate.
In a preferred embodiment of the invention, described complete culture solution is: take DMEM/F12 nutrient solution as basis, comprise that final concentration is respectively 16.7% foetal calf serum FBS, 0.5 ‰ beta-mercaptoethanols, 50 μ g/mL N-acetyl glucose osamines, 50 μ g/mL Xylo-Mucines, 10 μ g/L people FGF-basic, 5 μ g/L people EGF, 1 μ g/L people HGF, 100IU/mL penicillin and 100 μ g/mL Streptomycin sulphates.
The invention has the beneficial effects as follows:
1, muscles of Pseudosciaena crocea clone of the present invention can continuous passage, goes down to posterity for 40 generations so far, and a large amount of muscles of Pseudosciaena crocea clone can be provided, for pathogenic characteristic research, vaccine development, functional gene research and breeding research etc.
2, strong, the clone good stability that builds of the construction process of muscles of Pseudosciaena crocea clone of the present invention repeatability;
3, the cell proterties of muscles of Pseudosciaena crocea clone of the present invention is good.For inoblast, present cell space fusiformis or sealene triangle, the projection of the protruding several varying lengths of kytoplasm, be arranged in radial, vortex shape.Divide vigorously, the generation time is short, easy to digest, and adherent rate is good, resistance to hunger;
4, the complete culture solution that construction process of the present invention is used comprises DMEM/F12 nutrient solution, foetal calf serum FBS, beta-mercaptoethanol, N-acetyl glucose osamine, Xylo-Mucine, people FGF-basic, people EGF, people HGF, penicillin and Streptomycin sulphate, and DMEM/F12 nutrient solution and foetal calf serum FBS provide sufficient nutrient for Growth of Cells; Adding of beta-mercaptoethanol, N-acetyl glucose osamine, Xylo-Mucine, people FGF-basic, people EGF and people HGF can cell stimulating activity, accelerate cell fission propagation, for cells in vitro, cultivate good buffer environment is provided simultaneously, can make cell when long-term cultivation, maintain stable pH; Penicillin and Streptomycin sulphate have strengthened antimicrobial spectrum, and when former culture, bacteria growing inhibiting, prevents cell contamination effectively especially.
Accompanying drawing explanation
Fig. 1 is the former culture of the muscles of Pseudosciaena crocea clone of the present invention microphotograph (50 *) of the 7th day;
Fig. 2 is the former culture of the muscles of Pseudosciaena crocea clone of the present invention microphotograph (50 *) of the 15th day;
Fig. 3 is the former culture of the muscles of Pseudosciaena crocea clone of the present invention microphotograph (50 *) of the 61st day;
Fig. 4 is that muscles of Pseudosciaena crocea clone of the present invention goes down to posterity and cultivates the microphotograph (50 *) in the 3rd generation;
Fig. 5 is that muscles of Pseudosciaena crocea clone of the present invention goes down to posterity and cultivates the microphotograph (50 *) in the 18th generation;
Fig. 6 is that muscles of Pseudosciaena crocea clone of the present invention goes down to posterity and cultivates the microphotograph (50 *) in the 37th generation;
Fig. 7 is that muscles of Pseudosciaena crocea clone of the present invention goes down to posterity and cultivates the microphotograph (50 *) in the 51st generation;
Fig. 8 is the growth curve that muscles of Pseudosciaena crocea clone of the present invention goes down to posterity under ratio in difference;
Fig. 9 is the microphotograph (50 *) after the frozen rear recovery 24h of muscles of Pseudosciaena crocea clone of the present invention;
Figure 10 is the microphotograph (50 *) after the frozen rear recovery 72h of muscles of Pseudosciaena crocea clone of the present invention;
Figure 11 is the microphotograph (50 *) going down to posterity again after muscles of Pseudosciaena crocea clone cryopreservation resuscitation of the present invention
Figure 12 is the chromosome map (100 *) of muscles of Pseudosciaena crocea clone of the present invention;
Figure 13 is the chromosome number distribution plan of muscles of Pseudosciaena crocea clone of the present invention;
Figure 14 is the hungry microphotograph (50 *) of processing after 30 days of muscles of Pseudosciaena crocea clone of the present invention;
Figure 15 changes the complete culture solution microphotograph of 36 hours (50 *) after the hungry processing of muscles of Pseudosciaena crocea clone of the present invention;
Figure 16 is the muscles of Pseudosciaena crocea clone transfection pEGFP-N1 plasmid Photomicrograph of 36 hours of the present invention (100 *);
Figure 17 is the muscles of Pseudosciaena crocea clone transfection Oct4-pEGFP-N1 recombinant vectors microphotograph of 36 hours of the present invention (100 *).
Embodiment
By embodiment, by reference to the accompanying drawings technical scheme of the present invention is further detailed and is described below.
Solution preparation
D-Hanks solution: get 0.4g KCl, 0.06g KH
2pO
4, 8.0g NaCl, 0.35g NaHCO
3, 0.132g NaH
2pO
412H
2o is settled to 1L, in 121 ℃ of 20min autoclavings, and 4 ℃ of preservations.Separately get D-Glucose and be made into 200g/L stock solution, 121 ℃ of 20min autoclavings, store for cooling latter-20 ℃ long-term.During use, every L D-Hanks solution adds 5mL D-Glucose stock solution and 10mL100 * dual anti-solution (purchased from Corning Incorporated, this dual anti-100X concentrated solution is containing 10000U/mL penicillin and 10000 μ g/mL Streptomycin sulphates).
Contain dual anti-sterilizing seawater: get fresh seawater, in 121 ℃ of 20min autoclavings, during use, every 1L sterilizing seawater adds 10mL100 * dual anti-solution (purchased from Corning Incorporated, this dual anti-100X concentrated solution is containing 10000U/mL penicillin and 10000 μ g/mL Streptomycin sulphates).
Complete culture solution: get commercialization 500mL DMEM/F12 nutrient solution, then add final concentration to be respectively 16.7% foetal calf serum FBS, 0.5 ‰ beta-mercaptoethanols, 50 μ g/mL N-acetyl glucose osamines, 50 μ g/mL Xylo-Mucines, 10 μ g/L people FGF-basic, 5 μ g/L people EGF, 1 μ g/L people HGF, 100IU/mL penicillin and 100 μ g/mL Streptomycin sulphates.4 ℃ of preservations, be no more than 1 month duration of service.
10% glycerine frozen storing liquid: glycerine is in 121 ℃ of 20min autoclavings.The foetal calf serum of getting 10mL glycerine and 90mL mixes, 4 ℃ of preservations.
Colchicine stock solution: get above-mentioned configuration D-hanks solution, add 250mg colchicine, be settled to 125mL, fully dissolve, in super clean bench with 0.22 μ m syringe filters filtration sterilization.Stock solution concentration be 2mg/mL(100 *), after degerming, seal 4 ℃ of preservations.
Hypotonic solution: take 0.3g KCl and be dissolved in 100mL ultrapure water.Room temperature is permanently effective.
MTT stock solution: get above-mentioned configuration D-hanks solution, add 250mg MTT powder, fully dissolve, be settled to 50mL, in super clean bench with 0.22 μ m syringe filters filtration sterilization.Stock solution concentration is 5mg/mL, after degerming, with masking foil, wraps up lucifuge ,-20 ℃ of preservations.
Ka Nuoshi stationary liquid: acetic acid is mixed according to the ratio of 1:3 with methyl alcohol, and be placed on-20 ℃ of short-term preservations, prepare rear validity period and be no more than 1 day.
Giemsa dye liquor mother liquor: take 0.5g Giemsa dry powder, add 5mL glycerine, fully mill; Continue to add 28mL glycerine, with masking foil parcel, in 60 ℃ of heating 2h; Add 33mL methyl alcohol, mix.Mother liquor is deposited in Brown Glass Brown glass bottles and jars only, places after 3 weeks and uses, and room temperature is preserved for a long time.
Embodiment 1
The foundation of muscles of Pseudosciaena crocea clone of the present invention:
(1) because large yellow croaker belongs to Sciaenidae fish, extremely responsive to hypochlorite, therefore adopt containing dual anti-sterilizing seawater and replace hypochlorous acid thimerosal at body surface sterilisation step.By 7 months large, the Pseudosciaena crocea Fry of long 9-11cm is containing temporarily foster 1～2h in dual anti-sterilizing seawater.During end, splash into 3-4 and drip Eugenol, until fish body upset, belly upwards, to stimulate stress not behavior till.With sterile gauze piece, wipe fish skin mucus.To be soaked with the cotton balls wiping fish body surface 2 times of 75% alcohol.Fish body is moved into super clean bench, with dissecting utensil, take out muscle tissue, and put into and added in advance D-hanks culture dish rinsing 3-4 time.
(2) muscle tissue piece is intersected and shredded to 1mm with two No. 11 scalpels
3the small tissue blocks of size left and right.By the small tissue blocks cutting with containing dual anti-D-hanks solution rinsing 2 times, once with the nutrient solution rinsing containing serum somatomedin finally.Tissue block is inoculated in to 25cm
2in Tissue Culture Flask, absorb unnecessary nutrient solution.The body that overturns gently, makes it to be inverted.In 27 ℃ of constant temperature dry doubling 24h.Carefully add 5mL complete culture solution (containing dual anti-, somatomedin), right culturing bottle gently so that tissue block immerses in nutrient solution.In 27 ℃ of constant temperature culture, start former culture, change weekly complete culture solution once.
As shown in Figure 1 to Figure 3, muscles of Pseudosciaena crocea tissue has attached cell to migrate out tissue block (Fig. 1) on the 7th day after starting former culture, to the 15th day there is (Fig. 2) in existing small pieces cell cluster, occurs the stacking phenomenon of intensive cell (Fig. 3) to the 61st day around some tissue block.
(3) when the former culture attached cell of muscles of Pseudosciaena crocea clone fraction of coverage reaches 50-60% or when higher, obvious contact inhibition may appear in primary cell, now with trypsin digestion, start the cultivation of going down to posterity.Adopt classical cell dissociation step: remove the old nutrient solution in original culturing bottle; Add 5mLD-Hanks solution, clean 1 time to remove serum, the divalent-metal ion (this type of material can greatly affect tryptic digestion) in original fluid; Pour out above-mentioned D-Hanks solution, add the commercial trysinization liquid containing EDTA of 2.5mL0.25%, rock bottle, pancreatin solution is fully contacted with bottom cell, (this step should comparatively fast be carried out, otherwise easily causes digestion excessively to pour out trysinization liquid; While removing trysinization liquid, should leave the solution that enough makes flat maintenance moistening, otherwise the bottle end is prone to subregion, become dry and impact digestion); Shift out culturing bottle, under inverted microscope, observe, to most cells become circle remove adherent after, move into immediately super clean bench and add 5mL to contain serum free culture system liquid to stop digestion; Piping and druming, microscopy piping and druming effect, can repeat digestion once at most if residual cells is crossed; With the ratio of 1:2-10 by cell suspension inoculation to new culturing bottle, polishing nutrient solution is placed in 27 ℃ of constant temperature culture by culturing bottle to 5mL, goes down to posterity once every 2-8 days later.As shown in Fig. 4 to 7, muscles of Pseudosciaena crocea clone more stably presents fibre-like Polygons from 3 51 generations of generation to the.
Embodiment 2
Frozen and the recovery of the muscles of Pseudosciaena crocea clone of embodiment 1.
(1) frozen: to get one bottle of 75cm
2the vigorous confluent culture bottle of growth bottle at the bottom of muscles of Pseudosciaena crocea cell line cell, adopt trypsin digestion, centrifugal rear collecting cell precipitation, the cells frozen storing liquid that slowly adds 3mL to configure, and blow and beat gently cell, it is dispersed in cells frozen storing liquid, uses liquid-transfering gun that liquid is moved in cryopreservation tube.Cryopreservation tube is placed 1h at 4 ℃, then is placed in ice chest, ice chest is placed in to-80 ℃ and places 1 day, finally takes out cryopreservation tube and immerses in liquid nitrogen, can Long-term Cryopreservation.
(2) cryopreservation tube is taken out from liquid nitrogen, be placed on rapidly in the water-bath that regulates temperature (40 ℃), in the process of thawing cell, should constantly rock cryopreservation tube, its Quick uniform is thawed, until melt completely.The cell suspension of thawing is transferred to 25cm
2in culturing bottle centrifuge tube, to the complete culture solution of polishing 5mL in culturing bottle, in 27 ℃, 5%CO
2cultivate.The complete culture solution more renewing after 24h, continues to cultivate.
As shown in Figure 9, for the frozen rear recovery 24h adherent rate of muscles of Pseudosciaena crocea clone, reach 60%-80%, with frozen front cellular form no significant difference; At the bottom of after frozen rear recovery 72h, cell can cover with culturing bottle bottle as shown in figure 10; As Figure 11, the muscles of Pseudosciaena crocea cell after recovery can normally go down to posterity, and with frozen front cell and cryopreservation resuscitation after cellular form indifference.
Embodiment 3
The muscles of Pseudosciaena crocea clone difference of embodiment 1 go down to posterity impact and the population doubling time analysis of ratio growth curve:
The muscles of Pseudosciaena crocea clone of setting up from embodiment 1, choose that form is good, the cell of molecular marker for increased proliferation, had digestive transfer culture, is passaged to 25cm with 1:2,1:3,1:5 and 1:10 respectively
2tissue Culture Flask (to 4 culturing bottles, add respectively 2.5mL, 1.6mL, 1mL, 0.5mL cell suspension, then with nutrient solution by each bottle of solution total amount polishing to 5mL).From the 24h that goes down to posterity, every 24h, take pictures once, adopt classical 5 intersection sampling systems to be inverted microscopic photography statistics visual field observation cell count to culturing cell, get 5 point observation cell count summation mapping analysis for every group.
As shown in Figure 8, for muscles of Pseudosciaena crocea clone, with 1:2 or 1:3, go down to posterity by the mild phase of going through 1-2 days, enter logarithmic phase in the 3rd day, doubly, the 1:2 group Growth of Cells speed that goes down to posterity will obviously be better than 1:3 group to the 3-4 that cell number doubly increases to the 2nd day end; Go down to posterity group cell number and incubation time of 1:5 presents approximate proportional relationship, and cell number increases to be similar to same slope every day; And the higher multiple 1:10 that goes down to posterity can cause, cell proliferation is slow, stagnation.
Embodiment 4
The chromosome analysis of the muscles of Pseudosciaena crocea clone of embodiment 1:
Get the vigorous vigorous muscles of Pseudosciaena crocea cell of division of getting of division, be seeded to 75cm
2tissue Culture Flask, treats that cell enlargement is in logarithmic phase, and adding final concentration is the colchicine of 20 μ g/mL, and after 6-8h is cultivated in continuation, collecting cell obtains cell suspension.Cell suspension is moved to 15mL centrifuge tube, the centrifugal 10min of 1000g, sucking-off supernatant liquor, adds the hypotonic 30min of 4,mL3 ‰ KCl gently; Add the Ka Nuoshi stationary liquid of the fresh configuration precooling of 0.5mL to pre-fix 10min, the centrifugal 10min of 2000g; Get cell precipitation and add 0.5mL Ka Nuoshi stationary liquid, with the resuspended precipitation of liquid-transfering gun; Add again 1mL stationary liquid; 30cm height drips sheet to glass slide (in-20 ℃ of pre-treatment), and horizontal positioned is so that it thoroughly extends, and 65 ℃ dry; Then immerse containing the 10min that dyes in the dye vat of Giemsa dye liquor working fluid, distilled water rinses to remove surface of glass slide dregs, dry, resinene mounting, 1000 * oily sem observation counting.
As shown in Figure 12 and Figure 13, the chromosome analysis of large yellow croaker clone is shown to the caryogram of large yellow croaker clone all presents similar normal state and distributes: the 62% division phase cell chromosome number observing is 48; Chromosome number is distributed in (24-96 bar) between monoploid and tetraploid.
Embodiment 5
The hunger of the muscles of Pseudosciaena crocea clone of embodiment 1 is processed
The large yellow croaker cell of getting cell fraction of coverage 80%-100%, changes liquid with complete culture solution, and in 27 ℃, 5%CO2 cultivates, and finds that nutrient solution color becomes light yellowly by orange, illustrates that the nutritive substance in nutrient solution is consumed in a large number after 30d.Micro-Microscopic observation muscles of Pseudosciaena crocea cellular form, as shown in figure 14, removes adherent by a large amount of cells as seen and forms empty shape; Part cell, due to nutrient consumption cell generation shrinkage, is examined under a microscope refractivity and is strengthened; Cell still has more cell survival and form not to change.As shown in figure 15, after the fresh complete culture solution of supply again, the cell of surviving can rise in value again, divides still vigorously, and form does not change yet.
Embodiment 6
Muscles of Pseudosciaena crocea clone transfection pEGFP-N1 plasmid and the functional gene oct4 of embodiment 1
Forward and reverse primer of design large yellow croaker oct4 gene, amplification does not contain the orf of the large yellow croaker oct4 gene of terminator codon.Forward primer oct4F:TAC
gAGCTCgCCACCATGACGGAGAGACC and reverse primer oct4R:ACG
gAATTCtTCCAGTCAGGTGACTAACC, wherein GAGCTC is SacI restriction enzyme site, and GCCACC is for strengthening the Kozak sequence of expressing, and GAATTC is EcoRI restriction enzyme site, and T is the occupy-place base of avoiding three sub-phase shift mutations.With SacI and EcoRI restriction endonuclease, pEGFP-N1 plasmid is carried out to double digestion, and it is upper that the orf fragment of the large yellow croaker oct4 gene amplifying is linked to eukaryon expression plasmid pEGFP-N1, successfully constructs oct4-pEGFP-N1 recombinant plasmid.
Competence bacterial cell preparation: adopt bacillus coli DH 5 alpha bacterial classification, adopt classical Calcium Chloride Method to prepare competent cell, preserve for-80 ℃ long-term.
Plasmid transforms: get 1ng DNA solution and be placed in EP pipe, ice bath is placed.From-80 ℃ of refrigerators, take out 100 μ L competence bacterial suspensions, dissolve rapidly, add in DNA solution ice bath 10min.42 ℃, water-bath 2min.EP pipe is turned and cultivates 1h in 37 ℃ 220.Then the bacterium liquid after transforming is coated containing on antibiotic LB solid culture flat board.
Plasmid preparation: extract pEGFP-N1 plasmid and oct4-pEGFP-N1 recombinant plasmid ,-20 ℃ of preservations according to the large extraction reagent kit of HiPure Plasmid MaxiPrep Kit plasmid (TransGene company) specification sheets.
Transfectional cell: get the muscles of Pseudosciaena crocea cell line cell of vigorous growth, be inoculated in 12 orifice plates with 1:2,27 ℃, 5%CO2 cultivates.After cell fraction of coverage to 70%-80%, according to sofast transfection reagent (purchased from sun horse company) specification sheets, operate.27 ℃, 5%CO
2cultivate, 24h detects luciferase expression later.
Utilize after shuttle China-Sofast mediation plasmid DNA transfection muscles of Pseudosciaena crocea clone 24h, under fluorescent microscope, microscopy can detect green fluorescence, as shown in Figure 16 and Figure 17, pEGFP-N1 plasmid and-large yellow croaker functional gene oct4 has expression in this clone.After transfection 36h, still can observe green fluorescence.Illustrate that the muscles of Pseudosciaena crocea clone of setting up can adapt to the transfection experiment of foreign gene.
The above, be only preferred embodiment of the present invention, therefore can not limit according to this scope of the invention process, the equivalence done according to the scope of the claims of the present invention and description changes and modifies, and all should still belong in the scope that the present invention contains.
Claims (4)
1. a muscles of Pseudosciaena crocea clone, is characterized in that: this cell lies in and is deposited in Chinese Typical Representative culture collection center on October 31st, 2013, and preservation is registered on the books and is numbered: CCTCC NO:C2013158.
2. an establishment method for muscles of Pseudosciaena crocea clone claimed in claim 1, is characterized in that: comprise the steps:
(1) obtaining of muscles of Pseudosciaena crocea tissue: large by 7 months, the Pseudosciaena crocea Fry of long 9-11cm is supported temporarily 1-1.5 hour in containing the dual anti-sterilizing seawater of penicillin and Streptomycin sulphate, then splash into the Eugenol that accounts for sterilizing seawater volume 0.005-0.015%, until fish body upset, belly upward, to stimulator stress behavior till; With sterile gauze piece, wipe fish surface mucus, to soak behind spirituous cotton balls wiping fish surface, take out muscles of Pseudosciaena crocea tissue and be placed in containing penicillin and the dual anti-D-hanks liquid of Streptomycin sulphate;
(2) former culture: above-mentioned muscles of Pseudosciaena crocea tissue is shredded to 0.7-2.0mm
3small tissue blocks, with the rinsing of D-hanks liquid, finally with complete culture solution, wash; After rinsing, above-mentioned small tissue blocks is inoculated in Tissue Culture Flask, absorb unnecessary nutrient solution, 27 ℃ of constant temperature dry doublings of upset body 24 hours, add again complete culture solution to right body so that small tissue blocks immerses in nutrient solution, in 27 ℃ of constant temperature culture, start former culture, change weekly complete culture solution once;
(3) cultivation of going down to posterity: primary while being cultured to attached cell propagation to 50-60% fraction of coverage at least, remove old nutrient solution, and clean and remove residual serum and divalent-metal ion; Then with 0.25% trypsin digestion, go down to posterity and hanged cell, with 1:2-10, go down to posterity; Every 2-8 days, go down to posterity once later, described muscles of Pseudosciaena crocea Establishment of Cell Line success.
3. the establishment method of a kind of muscles of Pseudosciaena crocea clone as claimed in claim 2, is characterized in that: described complete culture solution comprises: DMEM/F12 nutrient solution, foetal calf serum FBS, beta-mercaptoethanol, N-acetyl glucose osamine, Xylo-Mucine, people FGF-basic, people EGF, people HGF, penicillin and Streptomycin sulphate.
4. the establishment method of a kind of muscles of Pseudosciaena crocea clone as claimed in claim 3, it is characterized in that: described complete culture solution is: take DMEM/F12 nutrient solution as basis, comprise that final concentration is respectively 16.7% foetal calf serum FBS, 0.5 ‰ beta-mercaptoethanols, 50 μ g/mL N-acetyl glucose osamines, 50 μ g/mL Xylo-Mucines, 10 μ g/L people FGF-basic, 5 μ g/L people EGF, 1 μ g/L people HGF, 100IU/mL penicillin and 100 μ g/mL Streptomycin sulphates.
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| CN104830760A (en) * | 2015-06-01 | 2015-08-12 | 中国海洋大学 | Turbot muscular cell line establishment method |
| CN108624553A (en) * | 2018-04-03 | 2018-10-09 | 华中农业大学 | A kind of high-efficiency transfection blueness Medaka muscle cell system |
| CN113637632A (en) * | 2021-07-20 | 2021-11-12 | 宁波大学 | Trachinotus ovatus muscle tissue 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 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN104830760B (en) * | 2015-06-01 | 2017-12-22 | 中国海洋大学 | A kind of method for building up of turbot muscle cell system |
| CN108624553A (en) * | 2018-04-03 | 2018-10-09 | 华中农业大学 | A kind of high-efficiency transfection blueness Medaka muscle cell system |
| CN113637632A (en) * | 2021-07-20 | 2021-11-12 | 宁波大学 | Trachinotus ovatus muscle tissue 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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