CN101914487B - Serum-free separating and culturing method for sheep embryo stem cell - Google Patents
Serum-free separating and culturing method for sheep embryo stem cell Download PDFInfo
- Publication number
- CN101914487B CN101914487B CN2010101001075A CN201010100107A CN101914487B CN 101914487 B CN101914487 B CN 101914487B CN 2010101001075 A CN2010101001075 A CN 2010101001075A CN 201010100107 A CN201010100107 A CN 201010100107A CN 101914487 B CN101914487 B CN 101914487B
- Authority
- CN
- China
- Prior art keywords
- sheep
- stem cell
- serum
- embryo stem
- esc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a serum-free separating and culturing method for a sheep embryo stem cell, comprising the following steps of: removing a zona pellucida of sheep blastula cultured in vitro with a Tyrode's Solution and then inoculating the sheep blastula to a serum-free culture solution of the sheep embryo stem cell and fixing by using a needle head; placing under the conditions that the temperature is 38.6DEG C and the saturated humidity is 5 percent CO2 for culturing; during the culturing, replacing the culture solution in a half quantity every 48 hours with the pH value of 6.8-7.2 andprimarily culturing for 7-9 days; and carrying out the transfer culture once according to the ratio of 1:2-1:4 by adopting a conventional mechanical method, that is to say, transferring the sheep embryo stem cell cultured in vitro to the 16th generation. The serum-free culture solution of the sheep embryo stem cell is prepared from D-MEM/F-12+80ng/mL bFGF+3muMCHIR 99021+10mu L/mL N2+20mu L/mL B27+10mu L/mL NEAA+2mM L-Glutamine+0.2mM beta-Mercaptoethanol. Bared embryos are fixed at the bottom of a culture dish by using a No.32 needle head so as to avoid the damage to the cells in the blastula, and a baked embryo trophocyte is stripped off. The preferable inoculating amount of the baked embryos is 45-60/groups. The method can ensure that the formation rate of primary colony of sheep embryo stem cells is improved to 33 percent (14/42).
Description
Technical field
The present invention relates to the biotechnology of embryonic stem cell, adopt mechanically peel and serum-free separation and Culture technological, for the production of Transgenic Sheep provides solid technology platform.
Background technology
Embryonic stem cell (Embryonic stem cell; ESC), be by before the Mammals implantation blastula stage inner cell mass (inner cell mass, ICM); After external specific culture environment is selected, is adapted to; The clone with maintenance undifferentiated state and unlimited multiplication capacity that obtains, its specific biological characteristics can make itself and ICM reintegrate, and participates in all processes of fetal development.Adopt the ESC clone technology, its integration efficiency is higher than the traditional core implantation technique far away, can produce the more animal with genetic homogeneity in a short time, removes progeny testing from, increases substantially the reproductive efficiency of breeding stock.Utilize ESC cells produce breeding transgenic livestock simultaneously; Will increase substantially feed conversion rate, promote growth of animal, improve the output of live-stock product; And can carry out the early stage selection of sex, polyembryony, product meat, product hair performance to livestock embryo at cell levels, shorten the cattle breeding time.
At present, in the research of sheep ESC, generally adopt conventional separation and Culture system both at home and abroad; By mouse embryo stem cell (Mouse Embryonic Stem Cell, mESC), human embryo stem cell (HumanEmbryonic Stem Cell, successful experience hESC); With the factor that influences mESC, hESC propagation, differentiation; Carry out organic assembling like feeder layer cells, conditioned medium, cytokine, growth factor, hormone, foetal calf serum and serum extract etc., according to mESC, hESC builds is standard, being applied directly to that sheep ESC builds is in the research; But up to now; Sheep ESC research does not obtain substantive breakthroughs as yet, and it is problem such as inefficiency that ubiquity is built, and the highest the 8th generation ESC that only obtained.About the correlative study of sheep embryo stem cell and the retrieval of the ESC algebraically that obtains report have: 1. flourishing in vain. biotechnology journal .2008 " influence that different culture systems separate, go down to posterity the sheep embryonic stem cell-like " the highest algebraically P8 of oESC; 2. Zhu, Sun etal.Zygote.2007 " Ovine (Ovis aries) blastula from an in vitro productionsystem and isolation of primary embryonic stem cells " the highest algebraically P3 of oESC; 3. Dattena, Chessa et al.Mol Reprod Dev.2006 " solation, culture, andcharacterization of embryonic cell lines from vitrified sheepblastocysts " the highest algebraically P5 of oESC; 4. Talbot, Rexroad et al.Mol Reprod Dev.1993 " Alkaline phosphatase staining of pig and sheep epiblast cells inculture " the highest algebraically P0 of oESC; 5. Handyside, Hooper et al.Development Genesand Evolution.1987 " Towards the isolation of embryonal stem cell linesfrom the sheep " the highest algebraically P1 of oESC.These have all greatly restricted research and the application of ESC technology in the sheep genetic breeding.The present invention in conjunction with at present domestic and international ESC research recent tendency, adopts mechanically peel and serum-free separating and culturing method as point of penetration, has stronger science, practicality and using value.
Summary of the invention
The objective of the invention is to: a kind of mechanically peel and serum-free separation and Culture technology that is used for sheep embryo stem cell (ESC) is provided; Make sheep ESC colony forming efficiency of former generation be increased to 33% (14/42); Vitro culture can reach for 16 generations, had expanded the ESC research field.
The objective of the invention is to realize like this: a kind of serum-free separating and culturing method that is used for sheep embryo stem cell; Sheep blastaea with vitro culture; After adopting Tyrode ' s Solution to remove zona pellucida, be inoculated in the homemade sheep embryo stem cell serum-free medium; With the fixing inner cell mass of syringe needle, place 38.6 ℃, 5%CO
2Cultivate under the saturated humidity condition, per 48 hours half amounts are changed nutrient solution in the cultivation, and pH is 6.8-7.2, and former being commissioned to train supported 7-9 days; Adopted the conventional mechanical method by 1 in every afterwards 3-4 days: 2-1: 4 go down to posterity once, and promptly the sheep embryo stem cell vitro culture can reach for 16 generations.
The serum-free separating and culturing method of described sheep embryo stem cell, the preparation of serum-free medium: D-MEM/F-12+80ng/mL bFGF+3 μ M CHIR99021+10 μ L/mL N2+20 μ L/mL B27+10 μ L/mL NEAA+2mM L-Glutamine+0.2mM β-Mercaptoethanol.
The serum-free separating and culturing method of described sheep embryo stem cell adopts No. 32 syringe needles that naked embryo is fixed in the petridish bottom, should avoid damaging the blastaea inner cell mass simultaneously, to greatest extent naked embryo trophocyte is peeled off as far as possible.
The serum-free separating and culturing method of described sheep embryo stem cell, naked embryonic breeding kind quantity is because of being advisable with 45-60 piece/group.
The serum-free separating and culturing method of described sheep embryo stem cell, this method can make the embryonic stem cell of sheep colony forming efficiency of former generation be increased to 33% (14/42) by 0% (0/129).
The serum-free separating and culturing method of described sheep embryo stem cell, selecting for use of medicament is the commercially available prod.
Research mechanism of the present invention comprises two sport technique segments of former pickup kind of sheep blastaea and sheep ESC serum-free culture:
The former pickup kind of sheep blastaea: the separation of traditional sheep blastaea inner cell mass; The main laboratory facilities such as immunosurgery modus operandi and enzyme process that adopt are peeled off the blastaea trophocyte; But because after blastaea long immunoreation of process and the enzyme processing operation; Be used for sheep ESC and build the inner cell mass that is, in various degree received the external source physical abuse, be efficient thereby influenced building of later stage sheep ESC; And can't essence solve the adherent key technical problem of blastaea inner cell mass, and this just finally causes sheep ESC colony forming efficiency of former generation lower.The present invention is the low main inducing of efficient from causing sheep ESC former generation to be built; Find through a large amount of laboratory proofings; Adopt No. 32 syringe needle; The naked embryo of removing zona pellucida is fixed in the petridish bottom (should avoids damaging the blastaea inner cell mass simultaneously as far as possible; And to greatest extent naked embryo trophocyte is peeled off), can significantly improve the former generation cloning efficiency of sheep blastaea inner cell mass under no feeder layer, serum free culture system, its sheep ESC colony forming efficiency of former generation is increased to 33% (14/42) by 0% (0/129) under this system.
The foundation of sheep ESC serum free culture system: combine international up-to-date ESC research tendency; Adopt serum free culture system; With reference to mouse embryo stem cell (Mouse Embryonic Stem Cell; MESC), human embryo stem cell (Human Embryonic Stem Cell; HESC) totipotency support mechanism; The factor that influences mESC, hESC propagation, differentiation is classified and combination; Successively start with from keeping mouse, the totipotent key signal regulatory pathway of human embryo stem cell LIF-STAT3 approach, MAPK-ERK approach, FGF approach, Wnt approach, the sheep ESC colony that former generation is obtained adds the factors (mLIF, hLIF, bFGF, EGF) from 2 kinds of nutrient solutions (DMEM/F12, KnockOut DMEM), 4 kinds of main added ingredientss (FBS, Serum Replacement, N2, B27), 4 kinds of micromolecular compounds (SU5402, PD0325901, CHIR99021), 4 kinds and has carried out a large amount of optimum combinations tests, and accumulative total is used more than 2100 piece of the external blastaea of sheep.Discover that when adding bFGF, when activating the FGF approach (Fig. 1 bFGF group), can significantly promote the self of sheep ESC, sheep ESC doubling time this moment is 2.0 days; In contrast; Add the FGF acceptor inhibitor, during blocking-up FGF approach (Fig. 1 SU group), the external doubling time of sheep ESC is extended for 3.7 days; Self receives obvious inhibition; Therefore after the screening contrast, the gained conclusion is that the regulatory pathway of sheep ESC self is the FGF approach, and this also provides scientific basis for further setting up sheep ESC serum free culture system.
Secondly, in the sheep ESC serum free culture system, the addition of bFGF has carried out series of contrast again for we.Discover that under this serum free culture system, sheep ESC is to the addition amount sexual demand (see figure 2) of bFGF: when adding 10ng/mL, 20ng/mL, 40ng/mL bFGF, sheep ESC self makes moderate progress, and is higher than the control group (see figure 2); When the bFGF addition was increased to 80ng/mL, 100ng/mL, sheep ESC self further was significantly improved, and the suitable (see figure 2) of the two effect.Therefore under we thought this serum free culture system, the optimum addition of bFGF should be at 80ng/mL.
Technique effect of the present invention:
(1) being used for that sheep ESC builds is the vitro culture blastaea of research, should be advisable with A level blastaea (inner cell mass big and obviously); B level, C level blastaea are difficult for forming former generation colony, and former generation colony forming efficiency on the low side; Adopt the time of Tyrode ' s Solution removal zona pellucida particularly important, should observe the deformation extent of zona pellucida constantly; Treatment time is long, is prone to cause segmentation cavity to subside, and peels off the trophocyte for follow-up mechanical process and causes certain difficulty;
(2) naked embryonic breeding kind quantity because of with 45-60 piece/group about be advisable; Inoculation quantity is low excessively, can cause sheep ESC colony forming efficiency of former generation on the low side because of paracrine approach disappearance;
(3) after the former pickup kind, should the next day observe the inner cell mass growing state, and in time adopt No. 32 syringe needles to peel off trophocyte residual in the seeded process; Residual trophocyte, its growth vigor is higher than the cell in the inner cell mass far away, and trophocyte's hyper-proliferative is to cause former generation colony forming efficiency on the low side, and the nutrient solution main inducing of meta-acid very easily;
(4) after the former pickup kind, palpus half amount was changed the ESC nutrient solution in per 48 hours; In time change nutrient solution, can effectively culture system pH be maintained between the 6.8-7.2, the long-term propagation of sheep ESC is had a very important role;
(5) each embryonic stem cell manipulation in vitro time is unsuitable long; Overlong time has irreversible damaging action to the long-term propagation of sheep ESC.
The present invention is the breach with the crucial regulatory factor that improves sheep blastaea inner cell mass colony forming efficiency of former generation and screening sheep ESC self; Put into practice with method through well-designed, adopt mechanically peel and serum-free separation and Culture technology, make sheep ESC colony forming efficiency of former generation reach 33% (14/42); Vitro culture can reach for 16 generations; This achievement in research has been expanded the ESC research field for through ESC technology production Transgenic Sheep solid strong technology platform being provided, and shows technical progress.
Description of drawings
To combine accompanying drawing that the present invention is described further below.
Accompanying drawing 1 is kept the effect growth figure of self to sheep ESC for the unlike signal path;
Like figure, shown in:
Control---control group, basic culture solution;
BFGF---add bFGF in the basic culture solution, activate the FGF approach;
SU---add SU5402 in the basic culture solution, suppress the FGF approach;
HLIF, mLIF---add hLIF, mLIF in the basic culture solution, activate the LIF-STAT3 approach;
PD---add PD0325901 in the basic culture solution, suppress the ERK approach.
Accompanying drawing 2 is that the bFGF approach is to sheep ESC self effect growth figure;
Accompanying drawing 3 is 7 days an amplification microgram (40x) of the former pickup kind of sheep ESC;
Accompanying drawing 4 is 7 days an amplification microgram (100x) of the former pickup kind of sheep ESC;
Accompanying drawing 5 is the sheep ESC inoculation amplification microgram (100x) in 9 days the 1st generation;
Accompanying drawing 6 is the sheep ESC inoculation amplification microgram (100x) in 12 days the 2nd generation;
Accompanying drawing 7 is the sheep ESC inoculation amplification microgram (100x) in 19 days the 5th generation;
Accompanying drawing 8 is the sheep ESC inoculation amplification microgram (100x) in 30 days the 8th generation;
Accompanying drawing 9 is sheep ESC inoculation 35 days the 10th generation amplification microgram (100x);
Accompanying drawing 10 is the sheep ESC inoculation amplification microgram (100x) in 54 days the 16th generation;
Accompanying drawing 11 is sheep the 9th a generation ESC alkaline phosphatase activities detection lug, red positive (100x);
Accompanying drawing 12 is sheep the 14th a generation ESC alkaline phosphatase activities detection lug, red positive (100x).
Embodiment
To combine just further explanation of invention of instance below.
Embodiment
Isolation cultivation method step: 42 pieces of the blastaeas that collection sheep vitro culture obtains; After adopting Tyrode ' sSolution to remove zona pellucida; Naked embryo is inoculated in the sheep ESC serum-free medium that this invention provides, and adopts No. 32 syringe needles that naked embryo is fixed in the petridish bottom, should avoid damaging the blastaea inner cell mass simultaneously as far as possible; And to greatest extent naked embryo trophocyte is peeled off, place 38.6 ℃, 5%CO afterwards
2Cultivate under the saturated humidity condition, former be commissioned to train support 7 days time (see Fig. 3,4 former generation colony), adopted the conventional mechanical method by 1 in per afterwards 4 days: 2-1: 4 go down to posterity once, per 48 hours half amount replacing nutrient solutions between during cultivation.And the 9th generation, the 14th generation sheep ESC that obtains carried out conventional alkaline phosphatase staining; Whether stem cell characteristic in its external long-term cultivation process lost judge (concrete detection method is seen Leukocyte AlkalinePhosphatase Kit explanation, and is red positive).
The preparation of reagent:
D-MEM/F-12(Invitrogen，11320033)，bFGF(Sigma，F0291-25UG)，N2Supplement(Invitrogen，17502048)，B27?Supplement(Invitrogen，17504044)，CHIR99021(Stemolecule，04-0004)，NEAA(Invitrogen，11140050)，L-Glutamine(Sigma，G8540-100G)，β-Mercaptoethanol(Amresco，0482-100ml)，Tyrode’s?Solution(Sigma，T1788-100ML)，Leukocyte?AlkalinePhosphatase?Kit(Sigma，86R)。(mentioned reagent is the commercially available prod)
Sheep ESC nutrient solution: D-MEM/F-12+80ng/mL bFGF+3 μ M CHIR99021+10 μ L/mLN2+20 μ L/mL B27+10 μ L/mL NEAA+2mM L-Glutamine+0.2mM β-Mercaptoethanol.
Experimental result:
Former pickup kind is after 7 days, separates former generation 14 pieces of colonies, former generation colony forming efficiency 33% (14/33) (see Fig. 3,4 former generation colony); Adopt sheep ESC serum-free medium to cultivate and obtain the 1st, 2,5,8,10,16 generation sheep ESC cells (seeing Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Figure 10) after 9 days, 12 days, 19 days, 30 days, 35 days, 54 days; The 9th generation, the 14th generation sheep ESC to obtaining carry out conventional alkaline phosphatase staining; Detected result is strong positive (seeing Figure 11, Figure 12); Show under this serum free culture system that the cell alkaline phosphatase activities that long-term cultivation obtains is higher, cell possesses the characteristic feature of embryonic stem cell.
Claims (3)
1. a serum-free separating and culturing method that is used for sheep embryo stem cell is characterized in that: with the sheep blastaea of vitro culture, after employing Tyrode ' s Solution removes zona pellucida, be inoculated in the sheep embryo stem cell serum-free medium; With the fixing inner cell mass of syringe needle, place 38.6 ℃, 5%CO
2Cultivate under the saturated humidity condition, per 48 hours half amounts are changed nutrient solution in the cultivation, and pH is 6.8-7.2, and former being commissioned to train supported 7-9 days; Adopted the conventional mechanical method by 1 in every afterwards 3-4 days: 2-1: 4 go down to posterity once, make the sheep embryo stem cell vitro culture reach for 16 generations;
Wherein: the preparation of serum-free medium: D-MEM/F-12+80ng/mL bFGF+3 μ M CHIR99021+10 μ L/mL N2+20 μ L/mL B27+10 μ L/mL NEAA+2mM L-Glutamine+0.2mM β-Mercaptoethanol;
Wherein: adopt No. 32 syringe needles that naked embryo is fixed in the petridish bottom, avoid damaging the blastaea inner cell mass, naked embryo trophocyte is peeled off.
2. according to the serum-free separating and culturing method of the described sheep embryo stem cell of claim 1, it is characterized in that: naked embryonic breeding kind quantity is 45-60 piece/group.
3. according to the serum-free separating and culturing method of the described sheep embryo stem cell of claim 1, it is characterized in that: this method can make the embryonic stem cell of sheep colony forming efficiency of former generation by 0%, 0/129, is increased to 33%, 14/42.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101001075A CN101914487B (en) | 2010-01-22 | 2010-01-22 | Serum-free separating and culturing method for sheep embryo stem cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101001075A CN101914487B (en) | 2010-01-22 | 2010-01-22 | Serum-free separating and culturing method for sheep embryo stem cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101914487A CN101914487A (en) | 2010-12-15 |
| CN101914487B true CN101914487B (en) | 2012-01-11 |
Family
ID=43322134
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010101001075A Expired - Fee Related CN101914487B (en) | 2010-01-22 | 2010-01-22 | Serum-free separating and culturing method for sheep embryo stem cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101914487B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127521B (en) * | 2010-12-25 | 2013-02-06 | 河南科技大学 | Method for removing mouse oocyte nuclei by adopting zona pellucida solution cavity method |
| CN102296047B (en) * | 2011-08-13 | 2013-03-13 | 新疆农垦科学院 | In vitro embryo production method by utilization of lamb superovulation oocytes |
| CN103275927A (en) * | 2013-06-20 | 2013-09-04 | 广东省实验动物监测所 | Embryonic stem cell and preparation method of feeder layer cell for cultivating embryonic stem cell |
| CN103555660B (en) * | 2013-09-09 | 2016-03-30 | 依科赛生物科技（太仓）有限公司 | A kind of embryonic stem cell serum free medium and application thereof |
| WO2019140260A1 (en) * | 2018-01-12 | 2019-07-18 | The Regents Of The University Of California | Efficient derivation of stable pluripotent bovine embryonic stem cells |
| CN111304155B (en) * | 2019-11-25 | 2021-08-20 | 内蒙古自治区农牧业科学院 | Separation culture method and culture solution for pluripotent stem cells in free state in sheep tissue |
-
2010
- 2010-01-22 CN CN2010101001075A patent/CN101914487B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN101914487A (en) | 2010-12-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101914487B (en) | Serum-free separating and culturing method for sheep embryo stem cell | |
| Goszczynski et al. | In vitro breeding: application of embryonic stem cells to animal production | |
| IS8618A (en) | Edible and cultured embryonic cells | |
| Wen et al. | The histone deacetylase inhibitor Scriptaid improves in vitro developmental competence of ovine somatic cell nuclear transferred embryos | |
| Gong et al. | Culture conditions and enzymatic passaging of bovine ESC-like cells | |
| Gu et al. | Rapid conversion of human ESCs into mouse ESC-like pluripotent state by optimizing culture conditions | |
| Kumar De et al. | Isolation and characterization of embryonic stem cell-like cells from in vitro produced goat (Capra hircus) embryos | |
| CN111269878B (en) | Special culture medium for converting human pluripotent stem cells into expanded pluripotent stem cells and application of special culture medium | |
| Zhao et al. | Derivation and characterization of ovine embryonic stem‐like cell lines in semi‐defined medium without feeder cells | |
| Ozawa et al. | Importance of culture conditions during the morula-to-blastocyst period on capacity of inner cell-mass cells of bovine blastocysts for establishment of self-renewing pluripotent cells | |
| Habibi et al. | Functional characterization of NANOG in goat pre-implantation embryonic development | |
| Zhang et al. | Rho kinase inhibitor Y-27632 and Accutase dramatically increase mouse embryonic stem cell derivation | |
| Zhang et al. | Research progress and application prospects of stable porcine pluripotent stem cells | |
| CN105062958A (en) | Composition for embryonic stem cell culture and application thereof | |
| CN104946581B (en) | A kind of special culture media and method for cultivating pig cytotrophoblast stem cells | |
| Goszczynski et al. | Embryonic stem cells as tools for in vitro gamete production in livestock | |
| Botigelli et al. | In vitro gametogenesis from embryonic stem cells in livestock species: recent advances, opportunities, and challenges to overcome | |
| Ko et al. | Embryonic stem cells as a potential source of gametes | |
| Qiu et al. | Effects of embryo aggregation and PXD101 on the in vitro development of mouse somatic cell nuclear transfer embryos | |
| Kwon et al. | Epiblast isolation by a new four stage method (peeling) from whole bovine cloned blastocysts | |
| CN106754656B (en) | Mouse or human totipotent stem cell quteASCs induction and culture establishment method | |
| CN109825468B (en) | Method for efficiently inducing fish pluripotent stem cells and induction medium used for method | |
| Niu et al. | Multipotent male germline stem cells (mGSCs) from neonate porcine testis | |
| CN111304157B (en) | Method for obtaining bovine initial state induced pluripotent stem cells | |
| Lee et al. | In vitro culture of chicken embryonic stem cell-like cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120111 Termination date: 20190122 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |