CN101798567B - Modified culture method for promoting cloned mouse embryonic development - Google Patents
Modified culture method for promoting cloned mouse embryonic development Download PDFInfo
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Abstract
The invention relates to a modified culture method for promoting cloned mouse embryonic development. The method is a continuous culture method-D culture method. The invention also relates to two culture systems capable of increasing the developmental rate of cloned mouse embryo, a method of using the culture systems for obtaining cloning animal and a method of using the culture systems for obtaining embryonic stem cells. By using the culture method of the invention, the developmental rate of cloned mouse embryo and the establishment efficiency of nuclear transfer of embryonic stem cells are increased, thus paving a new way for using clone technology in the production and facilitating the further development of animal cloning technology.
Description
Technical field
The present invention relates to a kind of method that improves the cloned mouse embryonic development rate; This method is a kind of cultured continuously method-D culture method; The invention still further relates to two kinds of culture systems that improve the cloned mouse embryonic development rate; Use said culture system to obtain the method for somatic cell clone animal, and use said culture system to obtain the method for embryonic stem cell.
Background technology
In recent years, body-cell neucleus transplanting (SCNT) technology has obtained success in a lot of species, but the efficient of SCNT is still lower.Research thinks that the inefficiencies of somatic cell nuclear transfer technique (SCNT) not exclusively causes (Jouneau et al., 2003 by the donor nuclei reprogrammed to a great extent; Latham 2004).In order to improve reprogramming efficiency, people study and have attempted a lot of methods, comprise the activation opportunity of reconstructed embryo and the influence of DMSO (Wakayama et al.; 2001), the influence of division of cytoplasm suppressor factor, stoning and notes nuclear influence (the Wakayama et al. on opportunity; 2003); Electro fusion method (Ogura et al., 2000) and micromanipulative technique aspect comprise two-step approach (Wakayama et al., 1998) and single stage method (OSM) (the Zhou Q et al. of piezoelectric ceramics auxiliary (PEM); 2003), but all do not obtain significant effect.Consider that dna methylation and acetylation of histone all are the important epigenetic marks that influences reprogramming of somatic cells and clone embryos growth.The epigenetic modification that people have studied with pathology medicament change donorcells and embryo improves somatocyte by the ability of ooecium matter reprogrammed.Wherein at SAH (S-adenosyl homocysteine; S-adenosyl-homocysteine) and TSA (NSC 630176; Trichostatin A) test on shows; Can significantly improve the ectogenesis rate of clone embryos behind the SAH processing donorcells, and TSA handles donorcells or reconstructed embryo all can significantly improve cloning efficiency and reprogramming efficiency (Rybouchkin et al., 2006; Byeong-Gyun Jeon et al., 2008; Kishigami et al., 2006).But can these medicines especially Secure Application be still waiting research to the clone embryos long-term effect in therapeutic cloning.
Discover that the vitro culture environment has significant effects to fetal development.So; Can perhaps optimizing vitro culture liquid improve clone embryos ectogenesis ability to change condition of in vitro culture? Chatot CL etc. are through discovering the retardance of mouse 2 cell stages; Glucose in the nutrient solution possibly suppress some crucial metabolic processes in early days in fetal development; And Stimulina is an energy derive (Chatot CL et al., 1989) first-selected in the early embryo development process.Retardance takes place in those that assert traditionally 2 cells when ectogenesis or embryo's difference of the mouse species that do not block also maybe relevant with the osmotic pressure of culture environment (Hadi etal., 2005).Kamjoo M etc. has compared the influence to normal spermatheca embryo apoptosis of mouse species and nutrient solution, and the result finds that the chemical substance in genetic origin and the nutrient solution is (Kamjoo et al., 2002) of equal importance to apoptosis.Each isolated area and molecular grouping in the somatocyte kytoplasm are reorientated (Gajewskiet al. according to the variation of composition in the culture environment; 2003); The epigenetic state can receive the influence of culture environment; For example the serum in the nutrient solution can change the epigenetic information relevant with genomic imprinting (Khosla et al., 2001).The expression of h19 gene can be changed (Doherty et al., 2000) by the vitro culture environment in the mouse zygote.Michele etc. discover that different with those normal fertilization zygotes, the blastaea rate of clone embryos and the regional distribution height of versatility mark Oct4mRNA in blastaea depend on the culture environment after the nuclear transplantation.After first cell cycle, the epigenetic modification of somatic cell nuclear is still continuing; The change that the reconstructed embryo center is reinvented helps or be unfavorable for setting up the zygotic gene program will depend on the special material (Michele Boiani et al., 2005) in the nutrient solution.Usefulness CZB such as Heindryckx, G1/G2 and KSOM/G2 cultivate discovering of clone and parthenogenetic embryo tire; Clone embryos and parthenogenetic embryo tire are very responsive to the vitro culture environment; Whether suitable ectogenesis (Heindryckx et al., 2001) of the nutrient solution of selecting with the remarkably influenced embryo.This shows; The vitro culture environment is very crucial to the embryo's in the various sources of mouse growth; This also points out us except the method for the raising cloning efficiency of above report, and studying and select the outer culture system of better embryoid body will be to influence the especially ectogenetic important factor of clone embryos of mice embryonic.
Therefore, need research and screen better placenta in vitro culture method and substratum improves the developmental rate of clone embryos, utilize new cultural method to can be the production of cloned animal and the acquisition of clone embryos stem cell brings new development opportunity.
Summary of the invention
In order to overcome the above problems; We use the combination of different culture liquid, respectively get advantage, seek more to promote the ectogenetic nutrient solution of cloned mouse embryo; We find a kind of cultured continuously method-D culture method first, can improve cloned mouse embryo's developmental rate significantly.
The invention provides a kind of method that improves the cloned mouse embryonic development rate; It is characterized in that; Said method is in-vitro cultivation method-D culture method of a kind of cloned mouse embryo, said method comprising the steps of: the reconstruct embryo is cultured to clone embryos and grows two-cell stage after activating in the substratum that does not contain EDTA and Stimulina; Change the KSOM substratum then into and cultivate, be cultured to clone embryos and grow blastaea.This substratum that does not contain EDTA and Stimulina is M16 substratum or CZB-EG (CZB that does not contain EDTA and Stimulina) substratum.The reconstruct embryo is through removing the karyomit(e) of nuclear transplantation acceptor, carries out the nuclear transplantation operation and obtains.The reconstruct embryo promptly can be described as clone embryos after activating.
The present invention relates to a kind of culture system that is used to improve the cloned mouse embryonic development rate; It is characterized in that said culture system constitutes by being used for after the reconstruct embryo activates clone embryos cultivated to grow the M16 substratum of two-cell stage and be used for that the clone embryos of said two-cell stage is cultivated the KSOM substratum of growing blastaea.
The invention still further relates to another kind and be used to improve the culture system of cloned mouse embryonic development rate; It is characterized in that; Said culture system is by being used for after the reconstruct embryo activates, clone embryos being cultivated the CZB that does not contain EDTA and Stimulina that grows two-cell stage; Be called CZB-EG, and be used for the clone embryos of said two-cell stage is cultivated the KSOM substratum formation of growing blastaea.
The present invention relates to a kind ofly be used to improve that NT-ESC (nuclear transfer embryo stem cell) builds is the method for efficient; It is characterized in that; The reconstruct embryo at M16 or do not contain and be cultured to clone embryos in the CZB substratum of EDTA and Stimulina and grow two-cell stage, changes the KSOM substratum then into and cultivates after activating; Be cultured to clone embryos and grow blastaea, set up the nuclear transfer embryo stem cell line.
The method of utilizing culture system of the present invention to obtain cloned animal comprises: after the reconstruct embryo activates in activation liquid; Carry out vitro culture with said culture system; Promptly at the M16 substratum or do not contain and be cultured to clone embryos in the CZB substratum of EDTA and Stimulina and grow two-cell stage, change the KSOM substratum then into and cultivate, grow blastaea during the stage; It is transplanted in the uterus of replace-conceive animal, obtains said cloned animal.
The method of utilizing culture system of the present invention to obtain the nuclear transfer embryo stem cell comprises: after the reconstruct embryo activates in activation liquid; Carry out vitro culture with said culture system; Promptly at the M16 substratum or do not contain and be cultured to clone embryos in the CZB substratum of EDTA and Stimulina and grow two-cell stage; Change the KSOM substratum then into and cultivate, grow blastaea during the stage, isolated nuclei transplanting embryo stem cell.
From the above mentioned, developmental rate and building of nuclear transfer embryo stem cell that the present invention has improved the cloned mouse embryo are efficient, have opened up new thinking for the applied research of clone technology on producing, and help the research of animal cloning; And help further developing of animal cloning.
Embodiment
Materials and methods
1 reagent
Unless stated otherwise, this research agents useful for same is Sigma (St.Louis, MO) Company products.
2 experimental animals
B6D2F1 mouse (C57BL/6 * DBA/2,8～12 weeks) is used to provide ovocyte and granulosa cell, and C57BL/6 (7～8 week) and the female mouse of ICR (8～12 week) provide zygote.The female mouse of 8～12 all ICR is also as the false pregnancy acceptor.The male mouse one of the ICR in 8～12 weeks is to be used for doing the public mouse of normal breeding, the 2nd, and ligation is used as joins the male mouse of false pregnancy.The male mouse of DBA, C57BL/6 and the female mouse of ICR and the male mouse of ICR are available from Beijing dimension tonneau China ltd.The B6D2F1 mouse obtains at this laboratory breeding, and all mouse are raised in cleaning level environment.
3 nutrient solutions
Each nutrient solution moity of table 1
Testing used nutrient solution mainly contains: CZB, M16, α MEM, KSOM.The ovocyte of collecting is temporarily cultivated in CZB, and nuclear transfer embryo is cultivated respectively at CZB, M16, α MEM, KSOM, M16+KSOM (D1), α MEM+KSOM.The reconstruct embryo activates the CZB+10 mM SrCl of liquid for no calcium ion
2+ 5 μ g/ml CB (cytochalasin B).Oocytes collection is used HEPES-CZB solution, and nuclear transplantation operates in the HEPES-CZB-CB solution to be carried out.
4 ovocytes, granulosa cell are collected
The female mouse of B6D2F1 and the female mouse abdominal injection of the ICR pregnant mare serum gonadotrop(h)in (PMSG) 10 (PMSG of unit in 8～12 ages in week; Ningbo), 48 hours pneumoretroperitoneum injection pregnancy urine extract 10 units (hCG, Ningbo); Inject back 15 hours disconnected necks and put to death mouse; Take off uterine tube, peel off the portion of expanding under the mirror, obtain ovarian cumulus ovocyte complex body (COC).(Costa Mesa CA) digests COC, and granulosa cell is separated with ovocyte, cleans secondary with HEPES-CZB for 300 U/ml, ICN Pharmaceuticals, and ovocyte moves in the CZB nutrient solution, is positioned over 37 ℃, 5%CO with Unidasa
2, cultivate in the incubator of saturated humidity.Granulosa cell is put in the nuclear transplantation operation liquid, get ovocyte after 20～30min carry out the nuclear transplantation operation.
The preparation of 5 normal fertilization ovum and body-cell neucleus transplanting (SCNT) clone embryos
After arranging with top program is ultra, C57BL/6 (7～8 week) and the female mouse of ICR (8～12 week) mate with DBA/2, the male mouse of ICR respectively; Examine bolt the next morning; The method of getting ovocyte with top description in 18～19 hours behind the injection hCG is got zygote, cultivates respectively in different culture liquid and observes developmental rate.
Single stage method (OSM) is all adopted in the body-cell neucleus transplanting operation, and the concrete operations step is with reference to report (Zhou et al., 2001 before us; Zhou et al., 2003).Reconstructed embryo is hatched the laggard line activating of 30min～60min in CZB, the SCNT reconstructed embryo is put among the no calcium ion CZB that contains 5 μ g/ml CB and handled 5～6 hours, and the clone embryos after the activation is incubated to be observed in the different culture liquid and the record developmental state.
6 fertilization embryo and clone embryos testing sequences
Table 2 fertilization embryo testing program
Table 3 clone embryos testing sequence
Fertilization embryo testing program is as shown in table 2.For example, for the D1 test group, the embryo that at first will be fertilized cultivates in the M16 substratum,, in the KSOM substratum, cultivates then to 2 cell stages until the fertilization fetal development, arrives blastaea until the fertilization fetal development.
The clone embryos testing sequence is as shown in table 3.For example,, at first clone embryos is cultivated in the M16 substratum behind the activation 0h-5h in activating liquid, grown to 2 cell stages, in the KSOM substratum, cultivate then, grow blastaea until clone embryos until clone embryos for the D1 test group.
The foundation of 7 NT-ES clones and stem cell are cultivated
The foundation of NT-ES clone (nuclear transfer embryo stem cell line) and cultivation are with reference to former report (Zhao et al., 2007).It is that the used white blood disease suppressor factor factor (LIF, leukaemia inhibitory factor) concentration is that (Chemicon, MA), and the used concentration of embryonic stem cell cultivation is 1000U to 2000 U that NT-ES clone is built.
8 embryo transfers
The SCNT blastaea is transplanted in 2.5 days the female mouse of the ICR uterus, and pregnant female mouse is c-section in 19.5 days after embryo transfer, and the birth offspring is through the female mouse replace-conceive of ICR.
9 data statistic analysis
Testing data adopts One-way ANOVA and the Fisher ' s Exact Test in SPSS 13.0 softwares to carry out data statistic analysis, and P in all analytical resultss＜0.05 is a significant difference, and P＜0.01 is that difference is extremely remarkable.
The result
The D culture method can significantly improve mouse somatic cell clone embryo's ectogenesis rate
In order to study cultural method to the ectogenetic influence of mice embryonic, we have compared the ectogenesis situation of B6D2F1 mouse SCNT embryo in the different culture method.
The result shows that different nutrient solutions are very big to the ectogenesis influence of clone embryos.Just evident difference occurs at 4 cell stages, wherein, do not had significant difference between embryo's 4 cell rates of cultivating among D1, α MEM, the α MEM/KSOM, and be significantly higher than CZB group (66.3%, P＜0.05), but not remarkable with M16 and KSOM group difference.Simultaneously, M16 and KSOM group does not have significant difference with the CZB group yet.But arrived morula, variation has taken place in situation, and the morula rate utmost point that D1 organizes is significantly higher than other each group (P＜0.01), and α MEM/KSOM group is only organized significant difference with M16, does not have significant difference between all the other each groups.In blastula stage, blastaea rate (62.3 ± 7.2%) utmost point of D1 group is significantly higher than other each group (P＜0.01).The result shows that the D culture method can significantly improve mouse somatic cell clone embryo's ectogenesis rate.
Table 4 clone embryos and the ectogenesis rate of fertilization embryo in different cultural methods
The different letter representation significant differences (P＜0.05) of shoulder motes in the same column
The influence that the D-method expires and grows SCNT (B6D2F1) embryo
For measuring the later stage developmental state of clone's blastaea that the D1 method cultivates, we transplant clone's blastaea that the D1 method is cultivated, and transplant and dissect 3 clone mouse in back 19.5 days; Body weight is respectively 2.10g, 1.32g, 1.54g; And placenta is respectively 0.27g; 0.20g, 0.11g, body weight and the placental weight of transplanting the back clone mouse that obtains with clone's blastaea of KOSM cultivation are similar.The fetus natality of D1 group almost is KOSM 2 times (1.5%vs.0.857%).The result shows that the D1 culture method can significantly improve mouse somatic cell clone embryo's the developmental rate that expires.
It is the influence of efficient that different cultural methods are built NT-ESCs
For further detecting the influence of D1 culture method to reprogramming of somatic cells, the building of nuclear transfer embryo stem cell line that we have compared clone's blastaea of different cultural methods acquisitions is efficient.The result shows that the degeneration blastaea ratio of D1 group is lower with the KSOM group than α MEM, and building is that efficient is more than the twice of α MEM group, also than KSOM group height.And the ratio of clone's blastaea that the D1 culture method obtains is significantly higher than other culture methods, therefore begins to calculate from reconstructed embryo, and it is efficient that the D1 culture method can significantly improve building of NT-ESCs.
Table 5: it is efficient that the NT-ESCs of different cultural methods builds
The different letter representation significant differences (P＜0.05) of shoulder motes in the same column
The influence that EDTA and Glutamine grow clone embryos in the nutrient solution
Other for example cultivate also that α MEM/CZB+KSOM can not improve SCNT embryo's ectogenesis rate for the ectogenesis of understanding why M16 and KSOM combination can raising clone embryos; We compare the moity of CZB and M16; The result finds do not have EDTA and Glutamine among the M16; Just make M16+KSOM promote SCNT embryo's ectogenesis just because of lacking these two compositions? Therefore we have removed EDTA or/and Glutamine in CZB; The SCNT embryo is cultivated; The result shows; The morula of the body-cell neucleus transplanting of cultivation in D1 culture system and CZB-Gln-EDTA (promptly having removed the CZB substratum of EDTA and Glutamine composition)+KSOM culture system and blastaea rate all are significantly higher than other each group, and the morula and the blastaea rate of cultivating the body-cell neucleus transplanting in CZB-Gln-EDTA (promptly having removed the CZB substratum of EDTA and Glutamine composition)+KSOM culture system are respectively 61.2% and 52.5%.Blastaea rate minimum (15.9%) in the CZB+KSOM group; Not remarkable with CZB-Gln (promptly having removed the CZB substratum of Glutamine composition)+KSOM group difference; But significantly be lower than CZB-EDTA (promptly having removed the CZB substratum of EDTA composition)+KSOM group, this shows, removed the growth that CZB and KSOM combined utilization behind the EDTA also can improve clone embryos to a certain extent; But still do not reach the effect of D1 group, have only the culture effect of removing behind EDTA and the Glutamine just to organize identical with D1.
In order to prove that further M16+KSOM promotes the SCNT ectogenesis to be because do not contain EDTA and Glutamine among the M16; We have added in M16 and unite KSOM again behind the Glutamine (Sigma) of EDTA (Sigma) and 1.0mM of 0.11mM the SCNT embryo is carried out vitro culture; The result finds that the M16+KSOM that has added EDTA and Glutamine has lost the ectogenetic ability of promotion SCNT embryo (18.6% (interpolation) vs.62.3% (not adding)).
Research shows, proves to add early stage (late periods 2 cell before) growth that EDTA and Glutamine are unfavorable for clone embryos in the nutrient solution.Exactly because M16+KSOM combination nutrient solution M16 does not contain EDTA and Glutamine has just possessed the ectogenetic ability of raising SCNT embryo.
Therefore, D culture method of the present invention comprises D1 culture method and D2 culture method; The D1 culture method promptly, is cultured to clone embryos and grows two-cell stage after activating the reconstruct embryo in the M16 substratum; Change the KSOM substratum then into and cultivate, be cultured to clone embryos and grow blastaea; The D2 culture method promptly, the reconstruct embryo is cultured to clone embryos and grows two-cell stage after activating in not containing the CZB substratum of YD 30 and Stimulina, change the KSOM substratum then into and cultivate, and is cultured to clone embryos and grows blastaea.
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Claims (6)
1. a method that is used to improve the cloned mouse embryonic development rate is characterized in that, the said method that is used to improve the cloned mouse embryonic development rate is a kind of cloned mouse embryo's an in-vitro cultivation method, i.e. D culture method said method comprising the steps of:
The reconstruct embryo is cultured to said clone embryos and grows two-cell stage after activating in the substratum that does not contain YD 30 and Stimulina, change the KSOM substratum then into and cultivate, and be cultured to said clone embryos and grow blastaea,
Wherein, the said substratum that does not contain YD 30 and Stimulina is selected from the M16 substratum and does not contain the CZB substratum of YD 30 and Stimulina.
2. culture system that is used to improve the cloned mouse embryonic development rate; It is characterized in that said culture system constitutes by being used for after the reconstruct embryo activates said clone embryos cultivated to grow the M16 substratum of two-cell stage and be used for that the clone embryos of said two-cell stage is cultivated the KSOM substratum of growing blastaea.
3. culture system that is used to improve the cloned mouse embryonic development rate; It is characterized in that; Said culture system is by being used for after the reconstruct embryo activates, said clone embryos being cultivated the CZB substratum that does not contain YD 30 and Stimulina of growing two-cell stage; Be called CZB-EG, and be used for the clone embryos of said two-cell stage is cultivated the KSOM substratum formation of growing blastaea.
4. one kind is used to improve that mouse nuclear transfer embryo stem cell builds is the method for efficient; It is characterized in that; The reconstruct embryo at the M16 substratum or do not contain and be cultured to clone embryos in the CZB substratum of YD 30 and Stimulina and grow two-cell stage, changes the KSOM substratum then into and cultivates after activating; Be cultured to said clone embryos and grow blastaea, set up the nuclear transfer embryo stem cell line.
5. method of utilizing claim 2 or 3 described culture systems to obtain mouse nuclear transfer embryo stem cells, said method comprises:
After the reconstruct embryo activates in activating liquid; Carry out vitro culture with said culture system; Promptly at the M16 substratum or do not contain and be cultured to clone embryos in the CZB substratum of YD 30 and Stimulina and grow two-cell stage; Change the KSOM substratum then into and cultivate, grow blastaea during the stage, isolated nuclei transplanting embryo stem cell.
6. according to claim 2 or the application of 3 described culture systems in obtaining nuclear transfer embryo stem cell, cloned animal, wherein, said nuclear transfer embryo stem cell is a mouse nuclear transfer embryo stem cell, and said cloned animal is the clone mouse.
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| CN106947733A (en) * | 2017-03-08 | 2017-07-14 | 阜阳师范学院 | A kind of extracorporeal culturing method for improving cloned mouse embryonic development rate |
| CN109609583A (en) * | 2018-12-04 | 2019-04-12 | 北京农学院 | Embryo's colouring method, the method and kit for judging Embryo viability |
| CN109749987B (en) * | 2019-01-18 | 2021-10-01 | 中国农业大学 | Method for preparing reconstructed embryo by suspension concussion co-culture and special composition thereof |
| CN112226404B (en) * | 2020-08-27 | 2023-09-05 | 吉林大学第一医院 | Culture medium composition and culture method for promoting animal embryo in-vitro development |
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