CN102277402A - Extraction method of nucleoprotein in animal cells - Google Patents
Extraction method of nucleoprotein in animal cells Download PDFInfo
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- CN102277402A CN102277402A CN2011102041375A CN201110204137A CN102277402A CN 102277402 A CN102277402 A CN 102277402A CN 2011102041375 A CN2011102041375 A CN 2011102041375A CN 201110204137 A CN201110204137 A CN 201110204137A CN 102277402 A CN102277402 A CN 102277402A
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Abstract
The invention discloses an extraction method of nucleoprotein in animal cells. In the method, cell penetrating peptide, exogenous nucleoprotein and enhanced green fluorescent protein are utilized to jointly construct a recombinant nucleoprotein expression vector; recombinant nucleoprotein is mediated to express in nuclei; after the recombinant nucleoprotein is stably expressed in the cells, the cells are treated by using an operation solution; the cells are collected and crushed with ultrasonic wave; after the cells are centrifuged to remove precipitate, the supernatant fluid of the cells is collected, filtered and sterilized, and added into a cell culture solution; and 4 hours later, the extracted fluorescence labeled exogenous recombinant nucleoprotein enters the nuclei through cytoplasm and is positioned in the nuclei. According to the method, the extracted nucleoprotein can play functions of protein, so that morphological character of the cell changes; and results prove that the method can easily, quickly and efficiently extract the nucleoprotein with bioactivity in the cells.
Description
[technical field]
The present invention relates to protein extracting method, relate in particular to a kind of zooblast kernel protein extracting method.
[background technology]
Proteinic preparation is a very elaboration, relates to multi-door subjects such as physics, chemistry and biology.Kinds of protein is a lot, differs greatly aspect physicochemical property, therefore all kinds of proteinic separation of fixed procedural application can not be arranged.Some form with solubility of protein in the animal material is present in body fluid such as blood, the urine, can directly separate through extracting.But other organism material combining form of some protein Chang Yiyu exists, and as nucleoprotein, this has brought big difficulty to mask work.Zooblast is made up of cytolemma, tenuigenin and nucleus three parts, and protein all exists in cytolemma, tenuigenin and nucleus in a large number, is bringing into play different physiological functions.Nucleus is the maincenter and the command centre of cell, expressed proteins in nucleus, be nucleoprotein, be unique a kind of albumen that is closely linked with the genetic material genome in three types albumen, nucleoprotein has only the homogenic group of proteic specific function of competence exertion that closely combines.Therefore, extract the bioactive nucleoprotein of maintenance difficulty maximum in three kinds of albumen types.
Genomic dna is the heavy-gravity thread-like molecule in the nucleus, and DNase I is the degrading enzyme of using always when extracting nucleoprotein removal genomic dna.The Chinese of DNase I is a deoxyribonuclease I, is a kind of endonuclease that is used to digest strand or double-stranded DNA.The ideal conditions of DNase I degrading genes group DNA is 37 ℃, and the primary condition that cell protein extracts requires to carry out under 0-4 ℃ cold environmental conditions, purpose is in order to prevent that cell protein from degrading fast, and therefore, the extraction of nucleoprotein will take into account two different temperature condition and be difficult to carry out.At present, the extraction of nucleoprotein is often adopted protein denaturant to make the nucleus protein denaturation and is split away off from genome.But the nucleoprotein that this method is extracted can only be used for proteinic routine analysis such as protein blot, protein electrophoresis, co-immunoprecipitation and detect because protein denaturation does not have the biologic activity of nucleoprotein.In addition, owing to contain protein denaturant in the protein liquid that extracts, the protein denaturant of trace can cause the death of culturing cell in the protein extract, therefore can not add to and carry out the protein function application in the cell culture fluid.
[summary of the invention]
The technical problem to be solved in the present invention provides a kind of zooblast kernel protein extracting method.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is that a kind of zooblast kernel protein extracting method may further comprise the steps:
(1) expression of nucleoprotein
Cultivate the 293T cell that goes down to posterity with the sugared DMEM nutrient solution of height, select the good 293T cell of fast growth, growth conditions as the nucleoprotein express cell through repeatedly cultivating the back of going down to posterity; When treating that the 293T cell grows into logarithmic phase; Adopt the mediation of calcium phosphate precipitation method Oct4, Sox2, Myc, Klf4 reorganization nucleoprotein expression vector at the 293T cell inner expression respectively;
(2) extraction of nucleoprotein
Fluorescent microscope is observed the expression of nucleoprotein in the 293T cell down, wait to recombinate nucleoprotein after cell inner expression is stable, choose the 293T cell that nucleoprotein is expressed respectively, remove the high sugared DMEM nutrient solution in the culture dish, add the high sugared DMEM nutrient solution that contains colchicine and handle cell, pending cellular form is collected 4 ℃ of placements in the centrifuge tube with the 293T cell together with nutrient solution after occurring changing; Take out the cell suspension under 4 ℃ of states, 4 ℃ of centrifugal supernatant liquors that go, high sugared DMEM nutrient solution re-suspended cell precipitation with 4 ℃ of precoolings, the centrifuge washing cell also removes supernatant liquor, with the high sugared DMEM nutrient solution of 4 ℃ of precoolings re-suspended cell precipitation again, add proteinase inhibitor simultaneously, blow and beat behind the mixing ultrasonication cell in the ice bath gently, 4 ℃ of centrifugal post precipitations that go are collected the supernatant liquid filtering degerming, and 4 ℃ of placements are standby.
The basic recombination structure of above-mentioned reorganization nucleoprotein expression vector is pcDNA3.1-cell-penetrating peptides-external source nucleoprotein-green fluorescent protein.
11 amino acid of above-mentioned cell-penetrating peptides consist of tyrosine-glycine-arginine-Methionin-Methionin-arginine-arginine-glutamine-Methionin-Methionin-Methionin.
(Cell penetrating peptide is to be positioned tenuigenin or nuclear aminoacid sequence after having permeates cell membranes or nuclear membrane CPP) to cell-penetrating peptides.Cell-penetrating peptides can carry exogenous protein or polypeptide enters in the cell, and does not influence and carry proteic biologic activity, is not subjected to the influence of cell type simultaneously.The basic structure that has the protein transduction effect in the aminoacid sequence of cell-penetrating peptides is the polypeptide fragment that is rich in basic aminoacids, and wherein arginine and lysine residue play an important role in the performance of cell-penetrating peptides function.
Green fluorescent protein (Enhanced green fluorescent protin, EGFP) be the most frequently used a kind of in the fluorescin family, from jellyfish, separate at first and obtain, luminophore mainly is the imidazole ring that forms by after Serine, tyrosine and the cyclisation of three amino acid processes of glycine, the oxidation, exciting at calcium ion does not need down other coenzyme can be separately or generation fluorescence when merging with other albumen, and fluorescence has high stability.Green fluorescent protein is owing to have the characteristic of autofluorescence, and the Chang Zuowei fluorescent mark is used widely in fields such as molecular biology, cytobiology and developmental biology.
The present invention adopts cell-penetrating peptides as the mediation material; the amino acid of its cell-penetrating peptides consists of tyrosine-glycine-arginine-Methionin-Methionin-arginine-arginine-glutamine-Methionin-Methionin-Methionin; amount to 11 amino-acid residues; connect cell-penetrating peptides; nucleoprotein and green fluorescent protein are formed reorganization nucleoprotein; Oct4 is adopted in experiment; Sox2; Myc; Klf4 reorganization nucleoprotein has been widely used in multipotent stem cells at present and has induced; the experiment by cell-penetrating peptides and green fluorescent protein verified the highly active protein matter that adopts new nucleoprotein extracting method can efficiently obtain native state; this method can be used in the proteic high efficiency extraction of a small amount of or a large amount of mass-producing nucleus; the nucleoprotein that extracts not only can be used for protein blot; protein electrophorese; conventional protein analysis such as co-immunoprecipitation detects, and carries out protein function applied analysis on the cell levels in the cell culture fluid but also can directly add to connect.
The invention has the beneficial effects as follows:
With respect to present other nucleoprotein extracting method, the present invention has other nucleoprotein extracting method not available two big characteristics:
(1) the crucial medicine of intracellular nucleic protein extraction is a colchicine, and colchicine is to extract from the liliaceous plant Colchicum autumnale as a kind of alkaloid at first, is low-cost conventional medicine, has been widely used in cell engineering at present.Colchicine makes karyomit(e) be stuck in metaphase by suppressing mitotic division.Be in the cell removal colchicine of holddown as the silk division after, cell then recovers normal mitotic division state again.Cell intracellular nucleoprotein when being in metaphase then splits away off from karyomit(e), therefore extracting nucleoprotein does not need to use and forbids the protein denaturant that uses in the cell culture fluid, has avoided Dnase I cell protein generation signs of degradation when 37 ℃ of digestion simultaneously yet.At present, conventional nucleoprotein extracting method all is to adopt protein denaturant or Dnase I on the market.
(2) the formed reorganization nucleoprotein of cell-penetrating peptides-nucleoprotein-green fluorescent protein can directly confirm the simple high efficiency of this nucleoprotein extracting method clearly, and the nucleoprotein that extracts has biological function, can be directly used in the functional verification of nucleoprotein.Simultaneously, green fluorescent protein can clearly directly be grasped nucleoprotein in intracellular state and location as cue mark.
From the above mentioned, it is efficiently simple that intracellular nucleic protein extracting method provided by the present invention has a leaching process, extracts visual result and understand, uses the cell material of minute quantity all to can be used for nucleoprotein and extract.Employed medicine of whole leaching process and reagent are all used in the cell cultures operation, and therefore, the nucleus albumen of extraction can directly add the biological function of performance nucleoprotein in the cell culture fluid to.
[description of drawings]
The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.
Fig. 1 is the goat fetal fibroblast form under the embodiment of the invention state of nature.
Fig. 2 is that the nucleoprotein of embodiment of the invention extraction limits the goat fetal fibroblast form after the factor is induced.
[embodiment]
1 test materials and method
1.1 main experiment material
The nucleoprotein express cell is the 293T cell;
11 amino acid of cell-penetrating peptides consist of tyrosine-glycine-arginine-Methionin-Methionin-arginine-arginine-glutamine-Methionin-Methionin-Methionin;
The basic recombination structure of reorganization nucleoprotein expression vector is pcDNA3.1-cell-penetrating peptides-external source nucleoprotein-green fluorescent protein;
The Tissue Culture Dish diameter is 60mm;
Cell culture fluid is high sugared DMEM nutrient solution;
1.2 main working method
1.2.1 the expression of nucleoprotein
Cultivate the 293T cell that goes down to posterity with the sugared DMEM nutrient solution of height, each 60mm culture dish 293T cell adds the high sugared DMEM nutrient solution of 5ml respectively, selects the good 293T cell of fast growth, growth conditions as the nucleoprotein express cell through repeatedly cultivating the back of going down to posterity.When treating that the 293T cell grows into confluent culture ware 80% left and right sides.Adopt the mediation of calcium phosphate precipitation method Oct 4, Sox2, Myc, Klf4 reorganization nucleoprotein expression vector at the 293T cell inner expression respectively.
1.2.2 the extraction of nucleoprotein
Fluorescent microscope is observed the expression of nucleoprotein in the 293T cell down behind the transfection 24h, choose the 293T cell of Oct4, Sox2, Myc, the expression of Klf4 nucleoprotein respectively, remove the high sugared DMEM nutrient solution in the culture dish, each 60mm culture dish 293T cell adds the high sugared DMEM nutrient solution 3ml that contains 1 μ g/ml colchicine preheating respectively, continue in 37 ℃ of incubators to cultivate, behind the 12h 293T cell is collected in the 15ml centrifuge tube together with nutrient solution, placed 8h for 4 ℃.Take out cell from 4 ℃ of refrigerators, under 4 ℃ of conditions at the centrifugal 3min of 1000rpm, abandoning supernatant, high sugared DMEM nutrient solution re-suspended cell precipitation with 4 ℃ of precoolings is transferred to cell suspension in the 2ml centrifuge tube, under 4 ℃ of conditions at the centrifugal 3min of 1000rpm, sop up supernatant liquor gently, add the high sugared DMEM nutrient solution 1ml of 4 ℃ of precoolings then, add 10 μ l proteinase inhibitor simultaneously, place standby gently behind the re-suspended cell in the ice bath.The cell suspension that ice bath is placed is put into ultrasonic cell disruption instrument together, ultrasonic time 3s, off time 5s, work number of times 30 times, ultrasonic power 80w is after the ultrasonication, with smudge cells suspension centrifugal 5min of 12000rpm under 4 ℃ of conditions, with the filter filtration sterilization of 0.22 μ m, 4 ℃ of placements are standby in Bechtop.
1.2.3 the functional verification of nucleoprotein
Cultivate the goat fetal fibroblast with the sugared DMEM nutrient solution of height, when treating that cell is in logarithmic phase, discard high sugared DMEM nutrient solution, get the high sugared DMEM nutrient solution of 200 μ l nucleoprotein liquid and 1800 μ l respectively and add in each 60mm Tissue Culture Dish the situation that observed and recorded nucleoprotein enters cell under the fluorescent microscope behind the 2h to.
Fetching the nucleoprotein equivalent mixed liquor that comes from Oct4, Sox2, Myc, Klf4 gene amounts to 200 μ l and mixes with the high sugared DMEM nutrient solution that fresh 1800 μ l contain 4 μ g/ml bFGF and 10U/ml LIF, add to then in the goat fetal fibroblast nutrient solution, 4h replenishes the high sugared DMEM nutrient solution that 2ml contains 4 μ g/ml bFGF and 10U/ml LIF, be replaced by the high sugared DMEM nutrient solution that contains 4 μ g/ml bFGF and 10U/ml LIF behind the 8h, repetitive operation is carried out 10d, the metamorphosis of microscopically observed and recorded goat fetal fibroblast continuously.
2 experimental results
2.1 nucleoprotein is appraised and decided the position in the 293T cell
Because nucleoprotein gene forms genetic recombinants with green fluorescence protein gene, the green fluorescent protein after the expression can accurately be located the expression position of external source nucleoprotein.After Oct4, Sox2, Myc, Klf4 nucleoprotein gene are transfected into 293T cell 24h under the calcium phosphate mediation, fluorescent microscope can be observed these nucleoprotein genes down and all express in the nucleus of 293T cell, the green fluorescent protein that mainly shows as the nucleoprotein connection is positioned at nucleus, the position of egfp expression is in full accord with the colour developing of nucleus specificity fluorescent dyestuff DAPI, does not have egfp expression in the kytoplasm of 293T cell.
2.2 the biological activity of nucleoprotein in fetal fibroblast
Cell-penetrating peptides can carry exogenous protein and enter in the cell, the position that green fluorescent protein can clear reflection albumen be arrived as cue mark.After the nucleoprotein liquid that extracts adds cell culture fluid 2h, fluorescent microscope can be observed a large amount of green fluorescent proteins of the interior gathering of tenuigenin of goat fetal fibroblast down, can see a small amount of green fluorescent protein in the nucleus, then can see a large amount of green fluorescent protein of the interior gathering of tenuigenin of goat fetal fibroblast behind the 4h, but assembled more green fluorescent protein in the nucleus, green fluorescent protein has passed through nuclear membrane and has entered in the nucleus, in nucleus, bring into play biological action by being positioned nucleus, in the nucleoprotein liquid that extracts, Oct4, Sox2, Myc, Klf4 combines with cell-penetrating peptides and green fluorescent protein and forms reorganization nucleoprotein, Oct4 under the effect of cell-penetrating peptides, Sox2, Myc, Klf4 nucleoprotein can enter performance biological activity in the nucleus, other cell protein composition in the protein liquid that extracts is not owing to can not enter in the cell with cell-penetrating peptides reorganization connection, still remain in the cell culture fluid, and be accompanied by the changing liquid of culturing cell and remove.
2.3 the mutagenic effect of nucleoprotein in fetal fibroblast
Oct4, Sox2, four kinds of nucleoprotein of Myc, Klf4 can be induced inoblast to produce inducing pluripotent stem cells and be brought into play mutagenic effect.The Oct4 that extracts, Sox2, Myc, four kinds of nucleoprotein liquid of Klf4 add the goat fetus to and become in the fiber nutrient solution, continuously after the interpolation effect 8d, in the goat fetal fibroblast of cultivating, can find that the variation of morphological specificity appears in the part cell, mainly show as the goat fetal fibroblast and be transformed into circle by fibrous form (Fig. 1), and iuntercellular is assembled formation cell cluster or cell clone mutually, present the peculiar cellular form of inducing pluripotent stem cells (Fig. 2), do not add Oct4, Sox2, Myc, the goat fetal fibroblast of four kinds of nucleoprotein liquid of Klf4 still keeps fibrous form under the same conditions, thereby show Oct4, Sox2, Myc, four kinds of nucleoprotein of Klf4 have had and have brought into play proteinic biologic activity, and four kinds of nucleoprotein actings in conjunction produce mutagenic effect.
Claims (3)
1. a zooblast kernel protein extracting method is characterized in that, may further comprise the steps:
(1) expression of nucleoprotein
Cultivate the 293T cell that goes down to posterity with the sugared DMEM nutrient solution of height, select the good 293T cell of fast growth, growth conditions as the nucleoprotein express cell through repeatedly cultivating the back of going down to posterity; When treating that the 293T cell grows into logarithmic phase; Adopt the mediation of calcium phosphate precipitation method Oct4, Sox2, Myc, Klf4 reorganization nucleoprotein expression vector at the 293T cell inner expression respectively;
(2) extraction of nucleoprotein
Fluorescent microscope is observed the expression of nucleoprotein in the 293T cell down, wait to recombinate nucleoprotein after cell inner expression is stable, choose the 293T cell that nucleoprotein is expressed respectively, remove the high sugared DMEM nutrient solution in the culture dish, add the high sugared DMEM nutrient solution that contains colchicine and handle cell, pending cellular form is collected 4 ℃ of placements in the centrifuge tube with the 293T cell together with nutrient solution after occurring changing; Take out the cell suspension under 4 ℃ of states, 4 ℃ of centrifugal supernatant liquors that go, high sugared DMEM nutrient solution re-suspended cell precipitation with 4 ℃ of precoolings, the centrifuge washing cell also removes supernatant liquor, with the high sugared DMEM nutrient solution of 4 ℃ of precoolings re-suspended cell precipitation again, add proteinase inhibitor simultaneously, blow and beat behind the mixing ultrasonication cell in the ice bath gently, 4 ℃ of centrifugal post precipitations that go are collected the supernatant liquid filtering degerming, and 4 ℃ of placements are standby.
2. zooblast kernel protein extracting method according to claim 1 is characterized in that, the basic recombination structure of described reorganization nucleoprotein expression vector is pcDNA3.1-cell-penetrating peptides-external source nucleoprotein-green fluorescent protein.
3. zooblast kernel protein extracting method according to claim 2, it is characterized in that 11 amino acid of described cell-penetrating peptides consist of tyrosine-glycine-arginine-Methionin-Methionin-arginine-arginine-glutamine-Methionin-Methionin-Methionin.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111518159A (en) * | 2020-03-31 | 2020-08-11 | 南昌大学 | Separation method of adherent cell nuclear protein |
| CN112779210A (en) * | 2020-03-18 | 2021-05-11 | 成都医学院 | Kit for extracting cytoplasm and cell nucleus proteins |
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| CN1207394A (en) * | 1997-08-04 | 1999-02-10 | 李京华 | Method for extracting chicken nucleic acid and nucleoprotein from chicken blood |
| JP2004016142A (en) * | 2002-06-18 | 2004-01-22 | Ls Corporation:Kk | Formulation containing collagen and water soluble decomposed product of nucleoprotein |
| WO2008028251A1 (en) * | 2006-09-07 | 2008-03-13 | The University Of Queensland | Dna-binding protein, nucleoprotein complex and method of use |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1207394A (en) * | 1997-08-04 | 1999-02-10 | 李京华 | Method for extracting chicken nucleic acid and nucleoprotein from chicken blood |
| JP2004016142A (en) * | 2002-06-18 | 2004-01-22 | Ls Corporation:Kk | Formulation containing collagen and water soluble decomposed product of nucleoprotein |
| WO2008028251A1 (en) * | 2006-09-07 | 2008-03-13 | The University Of Queensland | Dna-binding protein, nucleoprotein complex and method of use |
Non-Patent Citations (2)
| Title |
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| 罗艳等: "小鼠脾细胞核蛋白提取及核因子-kB活性的检测", 《创伤外科杂志》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112779210A (en) * | 2020-03-18 | 2021-05-11 | 成都医学院 | Kit for extracting cytoplasm and cell nucleus proteins |
| CN112779210B (en) * | 2020-03-18 | 2022-02-11 | 成都医学院 | Kit for extracting cytoplasm and cell nucleus proteins |
| CN111518159A (en) * | 2020-03-31 | 2020-08-11 | 南昌大学 | Separation method of adherent cell nuclear protein |
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