CN102732476A - Embryonic stem cell culture medium and application thereof - Google Patents
Embryonic stem cell culture medium and application thereof Download PDFInfo
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Abstract
The invention discloses an embryonic stem cell culture medium and application thereof. The embryonic stem cell culture medium comprises the following ingredients: a protein A containing an SOX2 protein, a protein B containing a POUV protein, a protein C containing a Nanog protein, a protein D containing a cLIN28 protein, a cell factor LIF, a cell factor SCF and a cell factor bFGF, wherein the SOX2 protein is specifically shown in Sequence 5; the POUV protein is specifically shown in Sequence 7; the Nanog protein is specifically shown in Sequence 9; and the cLIN28 protein is specifically shown in Sequence 11. When the embryonic stem cell culture medium provided by the invention is adopted to culture embryonic stem cells, the embryonic stem cells can be expanded in quantity for a long term and maintain good totipotency.
Description
Technical field
The present invention relates to a kind of embryonic stem cell substratum and application thereof.
Background technology
The cultivation of embryonic stem cell occupies extremely remarkable position always in biology medicine and pharmacology and Agricultural biotechnologies, recently because the active demand of transgenic technology and tissue regeneration medical science makes the research in this field demonstrate flourishing powerful situation.Set up and perfect mouse and human embryonic stem cell line, and dropped into practical study and used with treatment.But the fowl poultry kind embryonic stem cell line of genetically modified organism reactor drum institute active demand does not appear in the newspapers so far as yet.
Transgenic animal technology and transgenic animal pharmacy are one of the most popular, with fastest developing speed fields of Recent study.The purpose foreign gene imported in the animal body and with the animal gene group combine, along with the division and the propagation of cell, foreign gene in animal body expressing protein and animal from generation to generation between genetic stability.Transgenic animal have been broken between the kind in the natural propagation and have isolated; Making gene can kind be to flow between the far body of relation; The exchange and the reorganization of genetic material between the animal kind have been realized; This research that is not merely genetic material provides new means, has enriched the gene pool of species, and has enlarged the visual field of life science.1980; Gordon etc. breed transgenic mice first; At present except that transgenic mice; Transgene rabbit, Transgenic Sheep, transgenic pig, transgenic cattle, transgenic goat, transgenic chicken, transgenic rat, transgenic monkey etc. are bred successively, in fields such as Basic of Biology research, medical science, agricultural and biotechnology, all have high scientific research and economic worth.
The method of making transgenic animal mainly contains microinjection, retrovirus method, embryonic stem cell method, somatocyte implantation technique, adenovirus carrier method and sperm head and metastatic gene is total to injection etc.Embryonic stem cell is separated from early stage mice embryonic inner cell mass the earliest; In correct culture system can external go down to posterity for a long time and keep breeding at a high speed and keep the unlimited breeding of similar cancer cells with outwards, in, interior 3 well differentiated potential of germinal layer, can participate in vivo comprising that the growth of each histoorgan of reproductive tract forms mosaic.Through insert at random or homologous recombination with exogenous origin gene integrator in the genome of embryonic stem cell, obtain the transgenic cell of stably express, through resistance marker screening enrichment by cells transfected and set up embryonic stem cell line.Implant receiving in the stomatoblastula and will receiving stomatoblastula individual of normal development to this transgenic embryos stem cell with microinjection again to the transgenic animal that the intrauterine of receptor obtains.The embryonic stem cell mediated method is a transgenic animal technological line that has glamour.Compare with other several method, based on being that the embryonic stem cell mediated method of stablizing sophisticated embryonic stem cell line has the following advantages: (1) external source recombination is in the accurate integration that receives on the Autosome; (2) can, the embryo filter out suitable stable continuous cell line before implanting; (3) but pair cell carries out genetic modification, foreign gene regulating and expressing, operations such as gene self inactivation; (4) can set up the livestock and poultry species and the animal model of genetically deficient through the gene targeting gene knockout.But owing to lack sophisticated embryonic stem cell line, make this technology only be confined to the application on the mouse, seriously limit the performance of this modern technique advantage potential in numerous areas.
Induced multi-potent stem cells (iPS cell) is breakthrough progress in stem-cell research field in recent years; Its epoch making significance not only is for regenerative medicine and relevant treatment research a kind of abundant multipotent stem cells material source to be provided, and the more important thing is the stem cell versatility being kept and reprogramming of somatic cells dedifferentes Study on Mechanism has been introduced directly into gene regulating from the cell levels of inducing culture molecular level.
Bird is because its physiological property is uniquely occupied extremely significant status on medical animal model, life science fundamental research and drug manufacture.Poultry is human topmost meat and egg protein matter source always; The uterine tube of poultry is compared the value that mammiferous mammary gland has more becomes bio-reactor in addition; Poultry is easy to feeding and management and large-scale breeding; Egg albumen composition is simple and be easy to purifying, and the egg protein glycosylation more approaches the mankind.Obtain one stable, increase rapidly, have the bird embryonic stem cell line of many differentiation potentials, be that scientific research personnel institute urgently hopes and lays siege to always, also be bird transgenic field with other fundamental research and utilisation technology development strategic point need solution bottleneck.Existing bird embryonic stem cell culture system is based on the imitation to the embryonic stem cell culture system of mouse, mostly exist increases slowly, the shortcoming of poor stability, and experimental result is many can not repeat.And the special breeding physiology system of bird makes the embryonic stem cell of bird all fall far short with the model animals mouse from acquiring cultivation, makes to be difficult to be applied in the exploitation of bird embryonic stem cell culture system at very sophisticated embryonic stem cell culture system on the mouse.More than poultry, the research of the embryonic stem cell of other species also all is difficult to obtain the effect of mouse embryo stem cell, and the ripe embryonic stem cell culture system of visible existing mouse is among the embryonic stem cell of very difficult other animals of using is cultivated.This is because the gap between the species possibly be the intervention that on the regulated and control network of cell characteristics and versatility and self, is not suitable for the foreign cell factor owing to inner cell mass cell and blastoderm cells as the embryonic stem cell source on the other hand on the one hand.
Summary of the invention
The purpose of this invention is to provide a kind of embryonic stem cell substratum and application thereof.
The embryonic stem cell substratum that the present invention also provides comprises following component: contain the proteic albumen first of SOX2, contain the proteic albumen second of POUV, contain the proteic albumen of Nanog third, contain the proteic albumen fourth of cLIN28, cytokine LIF, cytokine SCF and cytokine bFGF.
SOX2 albumen shown in the sequence 5 that said albumen first is sequence table.POUV albumen shown in the sequence 7 that said albumen second is sequence table.Said albumen third is the Nanog albumen shown in the sequence 9 of sequence table.CLIN28 albumen shown in the sequence 11 that said albumen fourth is sequence table.
Said albumen first comprises following element: the l-arginine small peptide shown in the sequence 3 of SOX2 albumen shown in the sequence 5 of sequence table and sequence table.Said albumen second comprises following element: the l-arginine small peptide shown in the sequence 3 of POUV albumen shown in the sequence 7 of sequence table and sequence table.Said albumen third comprises following element: the l-arginine small peptide shown in the sequence 3 of Nanog albumen shown in the sequence 9 of sequence table and sequence table.Said albumen fourth comprises following element: the l-arginine small peptide shown in the sequence 3 of cLIN28 albumen shown in the sequence 11 of sequence table and sequence table.
Said embryonic stem cell substratum also can comprise following component: Sodium.alpha.-ketopropionate and chicken serum.
Said embryonic stem cell substratum specifically can be made up of DMEM substratum (like the sugared DMEM substratum of height) and solute; Said solute and the concentration in said embryonic stem cell goes down to posterity substratum thereof are following: Sodium.alpha.-ketopropionate 4-6mM (like 5mM), cytokine LIF 1-2 * 10
-2μ g/ml is (as 1.5 * 10
-2μ g/ml), cytokine SCF 0.5-1.5ng/ml (like 1ng/ml), cytokine bFGF 0.5-1.5ng/ml (like 1ng/ml), said albumen first 30-35mg/L (like 33.75mg/L), said albumen second 25-30mg/L (like 27.75mg/L), said albumen third 35-40mg/L (like 37.5mg/L), said albumen fourth 30-40mg/L (like 36mg/L or 31.5mg/L) and stripped chicken serum 80-120mL/L (like 100mL/L).
Said albumen first specifically can add with the form of protein liquid, and said protein liquid specifically can be the protein liquid that protein concentration is 0.45mg/ml, has specifically added 75mL in every liter of embryonic stem cell substratum.Said albumen second specifically can add with the form of protein liquid, and said protein liquid specifically can be the protein liquid that protein concentration is 0.37mg/ml, has specifically added 75mL in every liter of embryonic stem cell substratum.Said albumen third specifically can add with the form of protein liquid, and said protein liquid specifically can be the protein liquid that protein concentration is 0.5mg/ml, has specifically added 75mL in every liter of embryonic stem cell substratum.Said albumen fourth specifically can add with the form of protein liquid, and said protein liquid specifically can be the protein liquid that protein concentration is 0.48mg/ml or 0.42mg/ml, has specifically added 75mL in every liter of embryonic stem cell substratum.
The preparation method of the protein liquid of said albumen first is specific as follows:
(1) the SOX2 protein expressing plasmid is imported the 293FT cell, obtain reconstitution cell;
(2) said reconstitution cell was cultivated centrifugal collecting cell 12 hours for 37 ℃;
(3) cell of step (2) being collected carries out ultrasonic disruption (ultrasound parameter specifically can be: 450Hz, each ultrasonic 7s stops 9s, 44 times), collects the protein liquid of molecular weight less than 10KD then, is the proteic protein liquid of SOX2.
The preparation method of the protein liquid of said albumen first specifically also can be following:
(1) the SOX2 protein expressing plasmid is cut with Bgl II enzyme, obtained linearization plasmid;
(2) said linearization plasmid is passed through to import Chinese hamster ovary celI, obtain reconstitution cell.
(3) said reconstitution cell was cultivated 12 hours for 37 ℃, centrifugal (parameter of noncentricity specifically can be 3000rpm, 3min) collects supernatant, collects the protein liquid of molecular weight less than 10KD then, is the proteic protein liquid of SOX2.
The preparation method of the protein liquid of said albumen second is specific as follows:
(1) the POUV protein expressing plasmid is imported the 293FT cell, obtain reconstitution cell;
(2) said reconstitution cell was cultivated centrifugal collecting cell 12 hours for 37 ℃;
(3) cell of step (2) being collected carries out ultrasonic disruption (ultrasound parameter specifically can be: 450Hz, each ultrasonic 7s stops 9s, 44 times), collects the protein liquid of molecular weight less than 10KD then, is the proteic protein liquid of POUV.
The preparation method of the protein liquid of said albumen second specifically also can be following:
(1) the POUV protein expressing plasmid is cut with Bgl II enzyme, obtained linearization plasmid;
(2) said linearization plasmid is passed through to import Chinese hamster ovary celI, obtain reconstitution cell.
(3) said reconstitution cell was cultivated 12 hours for 37 ℃, centrifugal (parameter of noncentricity specifically can be 3000rpm, 3min) collects supernatant, adopts the protein liquid of molecular weight less than 10KD then, is the proteic protein liquid of POUV.
The preparation method of the protein liquid of said albumen third is specific as follows:
(1) the Nanog protein expressing plasmid is imported the 293FT cell, obtain reconstitution cell;
(2) said reconstitution cell was cultivated centrifugal collecting cell 12 hours for 37 ℃;
(3) cell of step (2) being collected carries out ultrasonic disruption (ultrasound parameter specifically can be: 450Hz, each ultrasonic 7s stops 9s, 44 times), collects the protein liquid of molecular weight less than 10KD then, is the proteic protein liquid of Nanog.
The preparation method of the protein liquid of said albumen third specifically also can be following:
(1) the Nanog protein expressing plasmid is cut with Bgl II enzyme, obtained linearization plasmid;
(2) said linearization plasmid is passed through to import Chinese hamster ovary celI, obtain reconstitution cell.
(3) said reconstitution cell was cultivated 12 hours for 37 ℃, centrifugal (parameter of noncentricity specifically can be 3000rpm, 3min) collects supernatant, collects the protein liquid of molecular weight less than 10KD then, is the proteic protein liquid of Nanog.
The preparation method of the protein liquid of said albumen fourth is specific as follows:
(1) the cLIN28 protein expressing plasmid is imported the 293FT cell, obtain reconstitution cell;
(2) said reconstitution cell was cultivated centrifugal collecting cell 12 hours for 37 ℃;
(3) cell of step (2) being collected carries out ultrasonic disruption (ultrasound parameter specifically can be: 450Hz, each ultrasonic 7s stops 9s, 44 times), collects the protein liquid of molecular weight less than 10KD then, is the proteic protein liquid of cLIN28.
The preparation method of the protein liquid of said albumen fourth specifically also can be following:
(1) the cLIN28 protein expressing plasmid is cut with Bgl II enzyme, obtained linearization plasmid;
(2) said linearization plasmid is passed through to import Chinese hamster ovary celI, obtain reconstitution cell.
(3) said reconstitution cell was cultivated 12 hours for 37 ℃, centrifugal (parameter of noncentricity specifically can be 3000rpm, 3min) collects supernatant, collects the protein liquid of molecular weight less than 10KD then, is the proteic protein liquid of cLIN28.
More than the preparation method of arbitrary said SOX2 protein expressing plasmid specific as follows: (SOX2 albumen is specifically shown in the sequence 5 of sequence table to insert SOX2 protein coding gene after removing terminator codon at the MCS (between EcoR I and BamH I restriction enzyme site) of recombinant plasmid pGSPF9R-IRES-EGFP; Its encoding sox is specifically shown in the sequence 6 of sequence table), obtain the SOX2 protein expressing plasmid.
More than the preparation method of arbitrary said POUV protein expressing plasmid specific as follows: (POUV albumen is specifically shown in the sequence 7 of sequence table to insert POUV protein coding gene after removing terminator codon at the MCS (between EcoR I and Kpn I restriction enzyme site) of recombinant plasmid pGSPF9R-IRES-EGFP; Its encoding sox is specifically shown in the sequence 8 of sequence table), obtain the POUV protein expressing plasmid.
More than the preparation method of arbitrary said Nanog protein expressing plasmid specific as follows: (Nanog albumen is specifically shown in the sequence 9 of sequence table to insert Nanog protein coding gene after removing terminator codon at the MCS (between EcoR I and BamH I restriction enzyme site) of recombinant plasmid pGSPF9R-IRES-EGFP; Its encoding sox is specifically shown in the sequence 10 of sequence table), obtain the Nanog protein expressing plasmid.
More than the preparation method of arbitrary said cLIN28 protein expressing plasmid specific as follows: (cLIN28 albumen is specifically shown in the sequence 11 of sequence table to insert cLIN28 protein coding gene after removing terminator codon at the MCS (between Kpn I and BamH I restriction enzyme site) of recombinant plasmid pGSPF9R-IRES-EGFP; Its encoding sox is specifically shown in the sequence 12 of sequence table), obtain the cLIN28 protein expressing plasmid.
More than the preparation of arbitrary said recombinant plasmid pGSPF9R-IRES-EGFP following: with the pIRES2-EGFP carrier is skeleton carrier; (the chicken growth hormone signal peptide is specifically shown in the sequence 1 of sequence table to insert the encoding sox of chicken growth hormone signal peptide in its MCS first (between Nhe I and Bgl II restriction enzyme site); Its encoding sox is specifically shown in the sequence 2 of sequence table); (the l-arginine small peptide is specifically shown in the sequence 3 of sequence table for the encoding sox of MCS second (between Sma I and BamH I restriction enzyme site) insertion l-arginine small peptide; Its encoding sox is specifically shown in the sequence 4 of sequence table), obtain recombinant plasmid pGSPF9R-IRES-EGFP; Said MCS first is positioned at the upper reaches of said MCS second.
More than arbitrary said embryonic stem cell substratum can be used for cultivating embryonic stem cell.Said embryonic stem cell specifically can be chicken embryonic stem cells (like the shouguang chicken embryonic stem cell).Said embryonic stem cell specifically can be blastoderm cells.
The present invention also protects a kind of method of cultivating embryonic stem cell, is the said embryonic stem cell of arbitrary described embryonic stem cell culture medium culturing more than the employing.Said embryonic stem cell specifically can be chicken embryonic stem cells (like the shouguang chicken embryonic stem cell).Said embryonic stem cell specifically can be blastoderm cells.
The present invention also provides and has contained the proteic albumen first of SOX2, contains the proteic albumen second of POUV, contains the proteic albumen of Nanog third, contains the proteic albumen fourth of cLIN28, cytokine LIF, cytokine SCF and the cytokine bFGF application in preparation embryonic stem cell substratum.
SOX2 albumen shown in the sequence 5 that said albumen first is sequence table.POUV albumen shown in the sequence 7 that said albumen second is sequence table.Said albumen third is the Nanog albumen shown in the sequence 9 of sequence table.CLIN28 albumen shown in the sequence 11 that said albumen fourth is sequence table.
Said albumen first comprises following element: the l-arginine small peptide shown in the sequence 3 of SOX2 albumen shown in the sequence 5 of sequence table and sequence table.Said albumen second comprises following element: the l-arginine small peptide shown in the sequence 3 of POUV albumen shown in the sequence 7 of sequence table and sequence table.Said albumen third comprises following element: the l-arginine small peptide shown in the sequence 3 of Nanog albumen shown in the sequence 9 of sequence table and sequence table.Said albumen fourth comprises following element: the l-arginine small peptide shown in the sequence 3 of cLIN28 albumen shown in the sequence 11 of sequence table and sequence table.
Said embryonic stem cell specifically can be chicken embryonic stem cells (like the shouguang chicken embryonic stem cell).Said embryonic stem cell specifically can be blastoderm cells.
Adopt embryonic stem cell substratum provided by the invention to carry out the cultivation of embryonic stem cell; Have if any advantage: can make long-term, a large amount of amplification of embryonic stem cell; And keep good totipotency (totipotency genes involved effective expression can form embryoid) and self ability (embryonic stem cell can go down to posterity for a long time); Easy and simple to handle, with low cost.The present invention can be used for external evoked differentiation, transgenic cell line preparation, stem cell nuclear transplantation, the nuclear transplantation of transgenic stem cell or mosaic preparation, can also be used for the application of multiple embryonic stem cell functions such as organizational project and cell therapy, has the major application prospect for biomedicine.
Description of drawings
Fig. 1 is the structural representation of the recombinant plasmid pGSPF9R-IRES-EGFP behind the insertion foreign gene.
Fig. 2 is P0 generation, P6 generation and the P7 of experimental group among the embodiment 3 surface marker detection for cell.
Fig. 3 is an embryonic stem cell totipotency related gene expression among the embodiment 3.
Fig. 4 is a formed embryoid body after cell passed through triploblastica gathering growth and broke up the 6th generation among the embodiment 3.
Fig. 5 is a chimeric photo among the embodiment 3.
Fig. 6 is the prolactin antagonist acceptor gene qualification result in each tissue of mosaic among the embodiment 3.
Fig. 7 is P0 generation, P6 generation and the P7 of experimental group among the embodiment 5 surface marker detection for cell.
Fig. 8 is an embryonic stem cell totipotency related gene expression among the embodiment 5.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
High sugared DMEM substratum: Gibco, article No. is 12491-015.Sodium.alpha.-ketopropionate: U.S. season biology ltd, article No. is A4859.Cytokine LIF:CANTA CRUZ, article No. are SC-4989.Cytokine SCF:Cell Signaling, article No. are #8925.Cytokine bFGF:Cell Sinaling, article No. are #8910.PIRES2-EGFP carrier: Ivitrogen, article No. are CB5159815.293FT cell: Ivitrogen, article No. are R700-07.Chinese hamster ovary celI: Ivitrogen, article No. are 2205-20-47.Shouguang chicken kind egg: China Agricultural University animal technical college education experiment field.Bai Laihang laying hen kind egg: China Agricultural University animal technical college education experiment field.
The preparation of embodiment 1, recombinant plasmid
One, the preparation of recombinant plasmid pGSPF9R-IRES-EGFP
With the pIRES2-EGFP carrier is skeleton carrier; (the chicken growth hormone signal peptide is shown in the sequence 1 of sequence table for the encoding sox of insertion chicken growth hormone signal peptide between its Nhe I and Bgl II restriction enzyme site; Its encoding sox is shown in the sequence 2 of sequence table); (the l-arginine small peptide is shown in the sequence 3 of sequence table, and its encoding sox is shown in the sequence 4 of sequence table for the encoding sox of insertion l-arginine small peptide between Sma I and the BamH I restriction enzyme site; Be abbreviated as 9 * Arg), obtain recombinant plasmid pGSPF9R-IRES-EGFP.Among the recombinant plasmid pGSPF9R-IRES-EGFP; It between the encoding sox of the encoding sox of chicken growth hormone signal peptide and l-arginine small peptide MCS; Can insert for foreign gene; In cell, express two albumen, one of them albumen is the fusion rotein that comprises chicken growth hormone signal peptide, foreign protein and l-arginine small peptide, and another albumen is EGFP albumen.When going out cell, the chicken growth hormone signal peptide is excised, and forms the fusion rotein that comprises foreign protein and l-arginine small peptide.
The structural representation of recombinant plasmid pGSPF9R-IRES-EGFP behind the insertion foreign gene is seen Fig. 1.
Two, the preparation of chicken source SOX2 protein expressing plasmid
(chicken source SOX2 albumen is shown in the sequence 5 of sequence table for chicken source SOX2 protein coding gene after inserting the removal terminator codon between the EcoR of recombinant plasmid pGSPF9R-IRES-EGFP I and the BamH I restriction enzyme site; Its encoding sox is shown in the sequence 6 of sequence table), obtain chicken source SOX2 protein expressing plasmid.Expression comprises the fusion rotein of following element: chicken source SOX2 albumen and l-arginine small peptide.
Three, the preparation of chicken source POUV protein expressing plasmid
(chicken source POUV albumen is shown in the sequence 7 of sequence table for chicken source POUV protein coding gene after inserting the removal terminator codon between the EcoR of recombinant plasmid pGSPF9R-IRES-EGFP I and the Kpn I restriction enzyme site; Its encoding sox is shown in the sequence 8 of sequence table), obtain chicken source POUV protein expressing plasmid.Expression comprises the fusion rotein of following element: chicken source POUV albumen and l-arginine small peptide.
Four, the preparation of chicken source Nanog protein expressing plasmid
(chicken source Nanog albumen is shown in the sequence 9 of sequence table for chicken source Nanog protein coding gene after inserting the removal terminator codon between the EcoR of recombinant plasmid pGSPF9R-IRES-EGFP I and the BamH I restriction enzyme site; Its encoding sox is shown in the sequence 10 of sequence table), obtain chicken source Nanog protein expressing plasmid.Expression comprises the fusion rotein of following element: chicken source Nanog albumen and l-arginine small peptide.
Five, the preparation of chicken source cLIN28 protein expressing plasmid
(chicken source cLIN28 albumen is shown in the sequence 11 of sequence table for chicken source cLIN28 protein coding gene after inserting the removal terminator codon between the Kpn of recombinant plasmid pGSPF9R-IRES-EGFP I and the BamH I restriction enzyme site; Its encoding sox is shown in the sequence 12 of sequence table), obtain chicken source cLIN28 protein expressing plasmid.Expression comprises the fusion rotein of following element: chicken source cLIN28 albumen and l-arginine small peptide.
The go down to posterity preparation of substratum of embodiment 2, embryonic stem cell
One, respectively the protein expressing plasmid of step 2 to the step 5 of embodiment 1 preparation is tested as follows:
1, protein expressing plasmid is passed through the calcium phosphate transfection method (reference of description calcium phosphate transfection method: NANCY HSIUNG, RAYMOND S.ROGINSKI, PAULA HENTHORN; OLIVER SMITHIES; RAJU KUCHERLAPATI, AND ARTHUR I.SKOULTCHI, Introduction and expression of a fetal human globin gene in mouse fibroblasts; MOLECULAR AND CELLULAR BIOLOGY; P.401-411) Apr.1982 imports in the 293FT cell, filters out the cell of expressing EGFP through observing fluorescence intensity after 72 hours.
2, the cell that step 1 screening is obtained was cultivated centrifugal collecting cell 12 hours for 37 ℃;
3, the cell of step 2 being collected carries out ultrasonic disruption (450Hz; Each ultrasonic 7s stops 9s; 44 times), adopt albumen evaporating column (Millipore UFC901024 Amicon Ultra-15 15ml/10KD, by specification operation) filtering and concentrating then; Collect the protein liquid of molecular weight, the freezing preservation of-80 degree less than 10KD.
Obtain chicken source SOX2 protein liquid (protein concentration is 0.45mg/ml), chicken source POUV protein liquid (protein concentration is 0.37mg/ml), chicken source Nanog protein liquid (protein concentration is 0.5mg/ml), chicken source cLIN28 protein liquid (protein concentration is 0.48mg/ml) respectively.
Replace expression plasmid to carry out step 1 recombinant plasmid pGSPF9R-IRES-EGFP, obtain reference protein liquid first to 3.
The 293FT cell carry out step 3, obtain reference protein liquid second.
Two, the chicken embryonic stem cells preparation of substratum of going down to posterity
The chicken embryonic stem cells substratum that goes down to posterity is made up of solute and high sugared DMEM substratum; The concentration of said solute in said chicken embryonic stem cells goes down to posterity substratum is following: Sodium.alpha.-ketopropionate 5mM, cytokine LIF 1.5 * 10
-2The chicken source Nanog protein liquid 75mL/L of the chicken source SOX2 protein liquid 75mL/L of μ g/ml, cytokine SCF 1ng/ml, cytokine bFGF 1ng/ml, step 1 preparation, the chicken source POUV protein liquid 75mL/L of step 1 preparation, step 1 preparation, the chicken source cLIN28 protein liquid 75mL/L and the chicken serum 100mL/L of step 1 preparation.
Three, contrast the preparation of the substratum first that goes down to posterity
Contrasting the substratum first that goes down to posterity is made up of solute and high sugared DMEM substratum; The concentration of said solute in the substratum first is gone down to posterity in said contrast is following: Sodium.alpha.-ketopropionate 5mM, cytokine LIF 1.5 * 10
-2The reference protein liquid first 300mL/L and the chicken serum 100mL/L of μ g/ml, cytokine SCF 1ng/ml, cytokine bFGF 1ng/ml, step 1 preparation.
Four, contrast the preparation of the substratum second that goes down to posterity
Contrasting the substratum second that goes down to posterity is made up of solute and high sugared DMEM substratum; The concentration of said solute in substratum second is gone down to posterity in said contrast is following: Sodium.alpha.-ketopropionate 5mM, cytokine LIF 1.5 * 10
-2The reference protein liquid second 300mL/L and the chicken serum 100mL/L of μ g/ml, cytokine SCF 1ng/ml, cytokine bFGF 1ng/ml, step 1 preparation.
Five, contrast the preparation of the substratum third that goes down to posterity
Contrasting the substratum third that goes down to posterity is made up of solute and high sugared DMEM substratum; The concentration of said solute in substratum third is gone down to posterity in said contrast is following: Sodium.alpha.-ketopropionate 5mM, cytokine LIF 1.5 * 10
-2The chicken source Nanog protein liquid 75mL/L of the chicken source SOX2 protein liquid 75mL/L of μ g/ml, cytokine SCF 1ng/ml, step 1 preparation, the chicken source POUV protein liquid 75mL/L of step 1 preparation, step 1 preparation, the chicken source cLIN28 protein liquid 75mL/L and the chicken serum 100mL/L of step 1 preparation.
Six, contrast the preparation of the substratum fourth that goes down to posterity
Contrasting the substratum fourth that goes down to posterity is made up of solute and high sugared DMEM substratum; The concentration of said solute in the substratum fourth is gone down to posterity in said contrast is following: the chicken source Nanog protein liquid 75mL/L of the chicken source SOX2 protein liquid 75mL/L of Sodium.alpha.-ketopropionate 5mM, cytokine SCF 1ng/ml, cytokine bFGF 1ng/ml, step 1 preparation, the chicken source POUV protein liquid 75mL/L of step 1 preparation, step 1 preparation, the chicken source cLIN28 protein liquid 75mL/L and the chicken serum 100mL/L of step 1 preparation.
The cultivation of embodiment 3, chicken embryonic stem cells
One, blastoderm cells obtains
Select the kind egg (shouguang chicken) of fresh hatching, get blastoderm cells.
Two, the cultivation of blastoderm cells
1, experimental group
With the chicken embryonic stem cells of the step 2 of the embodiment 2 preparation culture medium culturing blastoderm cells (P0 generation) that goes down to posterity; The observation of cell form; When intensive mountain peak shape cell mass appears in cell, digest and chicken embryonic stem cells that half amount more the renews substratum that goes down to posterity goes down to posterity, obtain P1 generation.Adopt aforesaid method to continue to go down to posterity, be followed successively by P2 generation, P3 generation, P4 generation, P5 generation, P6 generation, P7 generation.
2, control group first
Replace the chicken embryonic stem cells substratum that goes down to posterity to carry out the experiment of step 1 with the contrast of the step 3 of the embodiment 2 preparation substratum first that goes down to posterity.
3, control group second
Replace the chicken embryonic stem cells substratum that goes down to posterity to carry out the experiment of step 1 with the contrast of the step 4 of the embodiment 2 preparation substratum second that goes down to posterity.
4, control group third
Replace the chicken embryonic stem cells substratum that goes down to posterity to carry out the experiment of step 1 with the contrast of the step 5 of the embodiment 2 preparation substratum third that goes down to posterity.
5, control group fourth
Replace the chicken embryonic stem cells substratum that goes down to posterity to carry out the experiment of step 1 with the contrast of the step 6 of the embodiment 2 preparation substratum fourth that goes down to posterity.
Three, the evaluation of cytodifferentiation
1, P0 is for the identification of morphology of cell
In control group first and the control group second, P0 is following for cell form in culturing process: iuntercellular combines closely to be difficult to digest with digestive ferment, and cell has presented serious state of aggregation.
In the experimental group, P0 is still keeping the mountain peak shape for cellular form.
2, the generation time
Generation time is meant that the required time that can go down to posterity behind the mountain peak shape (cell density is at 1.5-2OD) appears in the blastoderm cells of cultivation.Each organize cell generation time (hour) see table 1.
Table 1 respectively organize cell generation time (hour) ("-" representative break up fully, do not have follow-up going down to posterity)
| P0 | P1 | P2 | P3 | P4 | P5 | P6 | P7 | |
| Experimental group | 20-24 | 20 | 20 | 22 | 24 | 24 | 48 | 48 |
| The control group first | 20-24 | 24 | - |
| Control group second | 20-24 | 24 | - | |||||
| Control group third | 20-24 | 24 | 60 | 96 | 132 | 180 | - | |
| The control group fourth | 20-24 | 24 | 60 | 96 | 132 | 180 | - |
3, the cell totipotency is identified
Get cell, carry out alkaline phosphatase staining and SSEA-1 and detect.
The method of alkaline phosphatase staining: cell is fixed 15 minutes with 4% ice-cold Paraformaldehyde 96, with the washing of PBS damping fluid, add alkaline phosphatase staining liquid then and (contain 100mM sodium-chlor, 5mM MgCl then
2, 1mg/ml NBT, the pH9.5 of 0.1mg/ml BCIP, 100mM Tris-HCl damping fluid) 37 ℃ of reactions 30 minutes, with PBS damping fluid washing back observation.APB is available from Ji Tai biotech company.NBT and BCIP are available from Promega.
The method that SSEA-1 detects: cell is fixed 15 minutes with 4% ice-cold Paraformaldehyde 96, and with the 1%BSA sealing, the anti-SSEA-1 monoclonal antibody of choosing then is (available from Cell Signaling then; By specification carries out 1:500 dilution) incubated at room 1 hour, with carrying out the fluorescent microscope detection after the washing of PBS damping fluid.
P0 generation, P6 generation and the P7 of experimental group sees Fig. 2 for the result of cell.P0 has stronger SEAP and SSEA-1 positive signal for cell.P6 has also shown the expression of SEAP and SSEA-1 preferably for cell.P7 is for cell, and mountain peak attenuation, SEAP and SSEA-1 dyeing shoal, and the cell of this moment is difficult to be dispersed into individual cells through the piping and druming of machinery, and iuntercellular connects comparatively tight, has explained that cytodifferentiation starts.Because cell was and paid the intensive state of aggregation this moment, was difficult to smash and continued and go down to posterity.
In control group first and the control group second, reach P1 for the time cell begin the differentiation.The P1 of control group first can observe cellular form for cell variation has taken place under the bright field, differentiation cells such as inoblast, epithelioid cell occurred being similar to, indicates that the blastoderm cells of this moment breaks up.
4, cell proliferation situation
(DOJINDO JC680) identifies viable cell quantity, i.e. cell proliferation situation to adopt the CCK-8 test kit.
Each is organized the propagation situation of cell and sees table 2.
Table 2 is respectively organized the propagation situation of cell
| P0 | P1 | P2 | P3 | P4 | P5 | P6 | P7 | |
| Experimental group | 1.417 | 1.396 | 2.767 | 2.635 | 1.734 | 1.633 | 1.286 | 1.097 |
| The control group first | 1.422 | 1.068 | 0.482 | 0.134 | - | - | - | - |
| Control group second | 1.442 | 1.081 | 0.427 | 0.149 | - | - | - | - |
| Control group third | 1.426 | 1.446 | 1.692 | 1.625 | 0.904 | 0.553 | - | - |
| The control group fourth | 1.442 | 1.471 | 1.706 | 1.668 | 0.999 | 0.589 | - | - |
The variation of each gene expression dose when 5, going down to posterity cultivation
Extract each total RNA for cell of experimental group, and reverse transcription being cDNA, is template with cDNA, adopts real-time fluorescence quantitative PCR to identify and keeps relevant each expression of gene level with the embryonic stem cell totipotency.
The primer that is used to identify the SOX2 gene is to as follows:
Upstream primer: 5 '-ATACCATGACGACCGGGGTA-3 ';
Downstream primer: 5 '-CGAGCTGGTCATGGAGTTGT-3 '.
The primer that is used to identify the Nanog gene is to as follows:
Upstream primer: 5 '-ATCCGTTCATGGCTGTGGAG-3 ';
Downstream primer: 5 '-CTGATGCCGTACAGGAGAGC-3 '.
The primer that is used to identify the POUV gene is to as follows:
Upstream primer: 5 '-GATGTTCAGCCAGACCACCA-3 ';
Downstream primer: 5 '-GGAAGAAGCTCTCCAGCGTT-3 '.
The primer that is used to identify the Lin28 gene is to as follows:
Upstream primer: 5 '-GGAGATTCACCCAAAGCCGA-3 ';
Downstream primer: 5 '-CCCGGATGGATTCCAAACCT-3 '.
The primer that is used to identify the c-Myc gene is to as follows:
Upstream primer: 5 '-CTACTTCGAGGAGGAGGAGGAGAA-3 ';
Downstream primer: 5 '-GATGATGATGGATTTGACGAAGGAT-3 '.
Be used to identify that internal control gene (b-actin) primer is to as follows:
Upstream primer: 5 '-CGTGACCTGACGGACTACCT-3 ';
Downstream primer: 5 '-GGGGCACCTGAACCTCTCATT-3 '.
Compare with internal control gene, the relative expression quantity of each gene is seen Fig. 3.
P5 is during for cell, and the expression amount of each gene (SOX2, Nanog, POUV, Lin28) is all identical with primary cell, and obvious variation does not take place, and has kept totipotency state preferably.P5 is during for cell, and tangible decline has appearred in Lin28, and reduction has in various degree also appearred in the expression of other associated transcription factors after this.
6, the evaluation of pluripotency differentiation
When identifying the embryonic stem cell totipotency, except that needs keep totipotency genes involved normal expression, also need measure cell differentiation capability in vitro and in vivo.
(1) external embryoid forms the mensuration of ability
The formation of triploblastica (in the one-tenth, in, outer three germinal layers) reflects that embryonic stem cell is at external many differentiation potentials.With experimental group P6 for cell transfer in the sugared DMEM substratum of height 37 ℃ cultivated 5 days, observe tridermic formation in the culturing process.
The result sees Fig. 4.Cultivate and begin to occur the spherical shape embryoid after 24 hours, in the middle of 48 hours, cavity occurs, formed comparatively typical embryoid structure, explain that it can be divided into the cell in three germinal layers sources, has shown totipotency preferably.
(2) mensuration of mosaic formation ability in the body
The P6 that gets experimental group is for the blastodisc of cell with the white Leghorn kind egg of 2000 (3ul) injection, and hatching is observed apparent chimeric situation after the hatching and PCR detects the chimeric situation of genome.
Chimeric formation can reflect many differentiation potentials of embryonic stem cell; In the embryonic stem cell of vitro culture can be divided in vivo; In, the cell of outer three germinal layers even gonadal cell, participate in form that chimeric histoorgan is grown in addition the F2 that produces donorcells for homozygote.Chimeric detection can be carried out from hair skin and genome detection both direction.The hair of shouguang chicken is that black and the hair chick of white Leghorn are yellow and form chicken and be white in color, and the prolactin antagonist acceptor gene of shouguang chicken is by the white long 21bp of Leghorn, more than 2 two contents that can satisfy the mosaic detection.
The photo of chick (mosaic) is seen Fig. 5, has black splotch.
The total RNA and the reverse transcription of extracting each tissue of chick are cDNA, are that template is carried out the PCR evaluation with cDNA.The primer that is used to detect the prolactin antagonist acceptor gene is concerning (for shouguang chicken, target sequence is 225bp, and for white Leghorn, target sequence is 201bp) as follows:
Upstream primer: 5 '-GGTGGGTGAAGAGACAAGGA-3 ';
Downstream primer: 5 '-TGCTGAGTATGGCTGGATGT-3 '.
Prolactin antagonist acceptor gene qualification result in each tissue of two chick (mosaic) is seen Fig. 6.
The go down to posterity preparation of substratum of embodiment 4, embryonic stem cell
One, respectively the protein expressing plasmid of step 2 to the step 5 of embodiment 1 preparation is tested as follows:
1, protein expressing plasmid is cut with Bgl II enzyme, obtained linearization plasmid;
2, the linearization plasmid with step 1 merges the (reference that electricity merges: Georges W.Bates, Sigrid A.Carle, William C.Piastuch through electricity; Linear DNA introduced into carrot protoplasts by electroporation undergoes ligation and recircularization; Plant Molecular Biology, June 1990, and Volume 14; Issue 6; Pp 899-908) imports Chinese hamster ovary celI,, obtain reconstitution cell through G418 screening (500ug/mL).
3, the cell that step 2 screening is obtained was cultivated 12 hours for 37 ℃; Centrifugal (3000rpm; 3min) collect supernatant, adopt albumen evaporating column (Millipore UFC901024 Amicon Ultra-15 15ml/10KD, by specification operation) filtering and concentrating then; Collect the protein liquid of molecular weight, the freezing preservation of-80 degree less than 10KD.
Obtain chicken source SOX2 protein liquid (protein concentration is 0.45mg/ml), chicken source POUV protein liquid (protein concentration is 0.37mg/ml), chicken source Nanog protein liquid (protein concentration is 0.5mg/ml), chicken source cLIN28 protein liquid (protein concentration is 0.42mg/ml) respectively.
Replace expression plasmid to carry out step 1 recombinant plasmid pGSPF9R-IRES-EGFP, obtain reference protein liquid first to 3.
The 293FT cell carry out step 3, obtain reference protein liquid second.
Two, the chicken embryonic stem cells preparation of substratum of going down to posterity
Three, contrast the preparation of the substratum first that goes down to posterity
Four, contrast the preparation of the substratum second that goes down to posterity
Five, contrast the preparation of the substratum third that goes down to posterity
Six, contrast the preparation of the substratum fourth that goes down to posterity
The cultivation of embodiment 5, chicken embryonic stem cells
One, blastoderm cells obtains
Select the kind egg (shouguang chicken) of fresh hatching, get blastoderm cells.
Two, the cultivation of blastoderm cells
1, experimental group
With the chicken embryonic stem cells of the step 2 of the embodiment 4 preparation culture medium culturing blastoderm cells (P0 generation) that goes down to posterity; The observation of cell form; When intensive mountain peak shape cell mass appears in cell, digest and chicken embryonic stem cells that half amount more the renews substratum that goes down to posterity goes down to posterity, obtain P1 generation.Adopt aforesaid method to continue to go down to posterity, be followed successively by P2 generation, P3 generation, P4 generation, P5 generation, P6 generation, P7 generation.
2, control group first
Replace the chicken embryonic stem cells substratum that goes down to posterity to carry out the experiment of step 1 with the contrast of the step 3 of the embodiment 4 preparation substratum first that goes down to posterity.
3, control group second
Replace the chicken embryonic stem cells substratum that goes down to posterity to carry out the experiment of step 1 with the contrast of the step 4 of the embodiment 4 preparation substratum second that goes down to posterity.
4, control group third
Replace the chicken embryonic stem cells substratum that goes down to posterity to carry out the experiment of step 1 with the contrast of the step 5 of the embodiment 4 preparation substratum third that goes down to posterity.
5, control group fourth
Replace the chicken embryonic stem cells substratum that goes down to posterity to carry out the experiment of step 1 with the contrast of the step 6 of the embodiment 4 preparation substratum fourth that goes down to posterity.
Three, the evaluation of cytodifferentiation
Method is all with embodiment 3.
The result of each step is following:
1, P0 is for the identification of morphology of cell
In control group first and the control group second, P0 is following for cell form in culturing process: iuntercellular combines closely to be difficult to digest with digestive ferment, and cell has presented serious state of aggregation.
In the experimental group, P0 is still keeping the mountain peak shape for cellular form.
2, the generation time
Each organize cell generation time (hour) see table 3.
Table 3 respectively organize cell generation time (hour) ("-" representative break up fully, do not have follow-up going down to posterity)
| P0 | P1 | P2 | P3 | P4 | P5 | P6 | P7 | |
| Experimental group | 20-24 | 20 | 20 | 22 | 24 | 24 | 48 | 48 |
| The control group first | 20-24 | 24 | - | |||||
| Control group second | 20-24 | 24 | - | |||||
| Control group third | 20-24 | 24 | 60 | 96 | 132 | 180 | - | |
| The control group fourth | 20-24 | 24 | 60 | 96 | 132 | 180 | - |
3, the cell totipotency is identified
In alkaline phosphatase staining and the SSEA-1 detection, P0 generation, P6 generation and the P7 of experimental group sees Fig. 7 for the result of cell.P0 has stronger SEAP and SSEA-1 positive signal for cell.P6 has also shown the expression of SEAP and SSEA-1 preferably for cell.P7 is for cell, and mountain peak attenuation, SEAP and SSEA-1 dyeing shoal, and the cell of this moment is difficult to be dispersed into individual cells through the piping and druming of machinery, and iuntercellular connects comparatively tight, has explained that cytodifferentiation starts.Because cell was and paid the intensive state of aggregation this moment, was difficult to smash and continued and go down to posterity.
In control group first and the control group second, reach P1 for the time cell begin the differentiation.The P1 of control group first can observe cellular form for cell variation has taken place under the bright field, differentiation cells such as inoblast, epithelioid cell occurred being similar to, indicates that the blastoderm cells of this moment breaks up.
4, cell proliferation situation
Each is organized the propagation situation of cell and sees table 4.
Table 4 is respectively organized the propagation situation of cell
| P0 | P1 | P2 | P3 | P4 | P5 | P6 | P7 | |
| Experimental group | 1.439 | 1.389 | 2.802 | 2.641 | 1.741 | 1.637 | 1.302 | 1.103 |
| The control group first | 1.429 | 1.071 | 0.479 | 0.129 | - | - | - | - |
| Control group second | 1.421 | 1.079 | 0.430 | 0.152 | - | - | - | - |
| Control group third | 1.429 | 1.435 | 1.674 | 1.619 | 0.925 | 0.548 | - | - |
| The control group fourth | 1.417 | 1.329 | 1.698 | 1.723 | 0.997 | 0.602 | - | - |
The variation of each gene expression dose when 5, going down to posterity cultivation
Compare with internal control gene, the relative expression quantity of each gene is seen Fig. 8.
P5 is during for cell, and the expression amount of each gene (SOX2, Nanog, POUV, Lin28) is all identical with primary cell, and obvious variation does not take place, and has kept totipotency state preferably.P5 is during for cell, and tangible decline has appearred in Lin28, and reduction has in various degree also appearred in the expression of other associated transcription factors after this.
Claims (10)
1. embryonic stem cell substratum comprises following component: contain the proteic albumen first of SOX2, contain the proteic albumen second of POUV, contain the proteic albumen of Nanog third, contain the proteic albumen fourth of cLIN28, cytokine LIF, cytokine SCF and cytokine bFGF.
2. embryonic stem cell substratum as claimed in claim 1 is characterized in that: the SOX2 albumen shown in the sequence 5 that said albumen first is sequence table; POUV albumen shown in the sequence 7 that said albumen second is sequence table; Said albumen third is the Nanog albumen shown in the sequence 9 of sequence table; CLIN28 albumen shown in the sequence 11 that said albumen fourth is sequence table.
3. embryonic stem cell substratum as claimed in claim 1 is characterized in that: said albumen first comprises following element: the l-arginine small peptide shown in the sequence 3 of SOX2 albumen shown in the sequence 5 of sequence table and sequence table; Said albumen second comprises following element: the l-arginine small peptide shown in the sequence 3 of POUV albumen shown in the sequence 7 of sequence table and sequence table; Said albumen third comprises following element: the l-arginine small peptide shown in the sequence 3 of Nanog albumen shown in the sequence 9 of sequence table and sequence table; Said albumen fourth comprises following element: the l-arginine small peptide shown in the sequence 3 of cLIN28 albumen shown in the sequence 11 of sequence table and sequence table.
4. like arbitrary described embryonic stem cell substratum in the claim 1 to 3, it is characterized in that: said embryonic stem cell substratum also comprises following component: Sodium.alpha.-ketopropionate and stripped chicken serum.
5. embryonic stem cell substratum as claimed in claim 4 is characterized in that: said embryonic stem cell substratum is made up of DMEM substratum and solute; Said solute and the concentration in said embryonic stem cell substratum thereof are following: Sodium.alpha.-ketopropionate 4-6mM, cytokine LIF 1-2 * 10
-2μ g/ml, cytokine SCF 0.5-1.5ng/ml, cytokine bFGF 0.5-1.5ng/ml, said albumen first 30-35mg/L, said albumen second 25-30mg/L, said albumen third 35-40mg/L, said albumen fourth 30-40mg/L and stripped chicken serum 80-120mL/L.
6. the application of arbitrary described embryonic stem cell substratum in cultivating embryonic stem cell in the claim 1 to 5.
7. a method of cultivating embryonic stem cell is to adopt arbitrary described embryonic stem cell substratum in the claim 1 to 5, cultivates said embryonic stem cell.
8. application as claimed in claim 6 or method as claimed in claim 7 is characterized in that: said embryonic stem cell is a blastoderm cells.
9. contain the proteic albumen first of SOX2, contain the proteic albumen second of POUV, contain the proteic albumen of Nanog third, contain the proteic albumen fourth of cLIN28, cytokine LIF, cytokine SCF and the cytokine bFGF application in preparation embryonic stem cell substratum.
10. application as claimed in claim 9 is characterized in that: said embryonic stem cell is a blastoderm cells.
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| CN103243070A (en) * | 2013-04-25 | 2013-08-14 | 刘小青 | Stem cell medium and application thereof |
| CN104152406A (en) * | 2014-08-15 | 2014-11-19 | 东北农业大学 | Preparation for enhancing differentiation capacity of chicken skeletal muscle myoblast and application thereof |
| CN106854646A (en) * | 2016-12-21 | 2017-06-16 | 南开大学 | A kind of mouse embryo stem cell of the low expression Lin28 of maintenance mouse embryo stem cell Naive states |
| CN114292809A (en) * | 2021-12-31 | 2022-04-08 | 吉林国健生命工程科学技术有限公司 | Culture medium containing chicken serum for in vitro cell culture and application of chicken blood in freshness |
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| CN102329771A (en) * | 2011-10-12 | 2012-01-25 | 佛山科学技术学院 | Method and special culture medium for subculturing chicken embryonic stem cells for long time |
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| 戴建明等: "鸡胚胎干细胞的分离和培养", 《中国畜牧杂志》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243070A (en) * | 2013-04-25 | 2013-08-14 | 刘小青 | Stem cell medium and application thereof |
| CN103243070B (en) * | 2013-04-25 | 2014-07-16 | 苏州佰通生物科技有限公司 | Stem cell medium and application thereof |
| CN104152406A (en) * | 2014-08-15 | 2014-11-19 | 东北农业大学 | Preparation for enhancing differentiation capacity of chicken skeletal muscle myoblast and application thereof |
| CN106854646A (en) * | 2016-12-21 | 2017-06-16 | 南开大学 | A kind of mouse embryo stem cell of the low expression Lin28 of maintenance mouse embryo stem cell Naive states |
| CN114292809A (en) * | 2021-12-31 | 2022-04-08 | 吉林国健生命工程科学技术有限公司 | Culture medium containing chicken serum for in vitro cell culture and application of chicken blood in freshness |
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