CN101851602A - Solution and method for culturing bovine somatic cell cloned embryos - Google Patents
Solution and method for culturing bovine somatic cell cloned embryos Download PDFInfo
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- CN101851602A CN101851602A CN200910021760A CN200910021760A CN101851602A CN 101851602 A CN101851602 A CN 101851602A CN 200910021760 A CN200910021760 A CN 200910021760A CN 200910021760 A CN200910021760 A CN 200910021760A CN 101851602 A CN101851602 A CN 101851602A
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Abstract
The invention provides a solution and method for culturing bovine somatic cell cloned embryos. The culture solution comprises merchant oviduct culture solution for bovine somatic cell cloned embryos (mSOF), and comprises activin A as well, wherein the concentration of the activin A is 20 to 80 ng/mL. The culture method comprises: culturing a bovine somatic-cell granular cell monolayer under conventional conditions for 3 days; adding the activin A in a concentration of 20 to 80 ng/mL to the merchant oviduct culture solution for bovine somatic cell cloned embryos (mSOF) from the fourth day; obtaining the solution for culturing bovine somatic cell cloned embryos; and using the solution for culturing bovine somatic cell cloned embryos for 5 days to obtain the bovine somatic cell cloned embryos. The solution and the method have the advantages of improving the 8/16 cell rate, blastula rate and hatchability of embryos and significantly raising the gene expression level of Na/K-ATPase, Glut-1, ActR II, Smad2 and the like, and are particularly suitable for application in the field of bovine somatic cell cloning.
Description
Technical field
The present invention relates to a kind of embryo medium and cultural method thereof, relate in particular to a kind of bovine somatic cells clone embryos nutrient solution and cultural method thereof.
Background technology
The essential vitro culture through certain hour of produced in vitro embryo filters out the measured embryo of matter and just can be transplanted to the acceptor intrauterine, so the vitro culture of bovine somatic cells clone embryos is one of key link that obtains the somatic cell clone ox.The embryo who grows in the body has the various unknown cytokines of parent reproductive tract excretory, and when vitro culture, the essential artificial various compositions that replenish, therefore has only the vitro culture environment very near uterine tube in the body and the normal development of intrauterine environment ability, to improve the clone embryos quality and quantity and to produce the healthy offspring.
At present at uterine tube nutrient solution (mSOF) (the Rorie R W of the ox embryo in vitro culturing liquid of being sold on the market for improvement, Lester T D, Miller G F, et al.Effects of protein source and coculture on bovine embryo development in synthetic oviductal fluid medium[J] .Theriogenology, 1994,42:385～395), its basal component mainly is CaCl
22H
2O, NaCL, KCL and KH
2PO
41.3mmol/L etc., the bovine somatic cells clone embryos is at 38.5 ℃, 5%CO
2, under the comparatively strict condition such as saturated humidity, cultivate on the granulosa cell individual layer and just can filter out embryo transfer after 8 days to the acceptor intrauterine.The culture medium matrix of even now originally can satisfy the vitro culture conditions needed, but this proportioning or such combination of agents make that embryo's 8/16 cell rate, blastaea rate, hatching rate are lower, and some important gene expression level is also relatively low.
Discovering that activator A is a member in the TGF-beta superfamily, is the interactional a kind of potential cytokine of mother-tire, can promote granulosa cell to produce steroid hormone, can promote the effect of FSH inductive aromatizing enzyme, thereby promote estrogenic secretion.Mainly, not only in early embryo development, play an important role by uterine epithelial cell and endometrial cell secretion, and very important to embryo's implantation.Activator A passes through to increase the reaction of the level increase of granulosa cell fsh receptor to FSH on the one hand; Stimulate granulosa cell to produce more aromatizing enzyme by FSH on the other hand, thereby promote estrogenic secretion.Still find no at present both at home and abroad the exploitation report of 8/16 cell rate that utilizes activator A to add the uterine tube nutrient solution to improve the bovine somatic cells clone embryos, blastaea rate, hatching rate etc., therefore, set up a kind of novel bovine somatic cells clone embryos nutrient solution that contains activator A is necessary very much.
Summary of the invention
The object of the present invention is to provide a kind of bovine somatic cells clone embryos nutrient solution, it has improved embryo's 8/16 cell rate, blastaea rate, hatching rate and Na/K-ATPase, Glut-1, gene expression doses such as ActRII, Smad2.
Technical solution of the present invention is:
A kind of bovine somatic cells clone embryos nutrient solution comprises commercially available bovine somatic cells clone embryos uterine tube nutrient solution (mSOF), and described bovine somatic cells clone embryos uterine tube nutrient solution (mSOF) comprises the CaCl of 1.5～2.5mmol/L
22H
2The KH of the NaCL of O, 100～112mmol/L, the KCL of 2.5～4.0mmol/L, 1.0～1.5mmol/L
2PO
4And the water of its surplus, its special expenditure is: also comprise activator A, the concentration of described activator A is 20～80ng/mL.
Above-mentioned bovine somatic cells clone embryos uterine tube nutrient solution (mSOF) comprises the CaCl of 2mmol/L
22H
2The KH of the NaCL of O, 107mmol/L, the KCL of 3.2mmol/L, 1.3mmol/L
2PO
4, the activator A of 20～80ng/mL and the water of its surplus.
The concentration of above-mentioned activator A is 40ng/mL the best.
Above-mentioned activator A is recombinant human activator A or ox activator A, is generally recombinant human activator A, because ox activator A generally is difficult to obtain.
A kind of cultural method that uses bovine somatic cells clone embryos nutrient solution of the present invention, its special character is that this cultural method comprises:
1) chooses bovine somatic cells granulosa cell individual layer, 38.5 ℃ of temperature, contain 5%CO
2And under the culture condition of saturated humidity, cultivated 3 days;
2) since the 4th day, adding concentration to commercially available bovine somatic cells clone embryos uterine tube nutrient solution (mSOF) is the activator A of 20～80ng/mL, obtains the described bovine somatic cells clone embryos of claim 1 nutrient solution.
3) use the described bovine somatic cells clone embryos of claim 1 nutrient solution 5 days again, promptly get the bovine somatic cells clone embryos.Nutrient solution provided by the present invention in use, nutrient solution is 38.5 ℃, 5%CO at culture condition
2, conventional culture condition such as saturated humidity.
Above-mentioned preceding 3 days cultivation need not changed liquid.
Above-mentioned back 5 days cultivation need be changed liquid once every other day.
Above-mentioned activator A needed 38.5 ℃ of temperature before commercially available bovine somatic cells clone embryos uterine tube nutrient solution (mSOF) adds, contains 5%CO
2And preheating 20～40 minutes under the condition of saturated humidity.
Above-mentioned activator A needed 38.5 ℃ of temperature before commercially available bovine somatic cells clone embryos uterine tube nutrient solution (mSOF) adds, contains 5%CO
2And 30 minutes the bests of preheating under the condition of saturated humidity.
The invention has the advantages that:
1, improves embryo's 8/16 cell rate, blastaea rate, hatching rate.
The present invention improves on the basis of original uterine tube nutrient solution (mSOF), has added activator A therein.When cultivating outside bovine embryo, the cell stage in 8-is generally blocked in fetal development, and at this moment period of maternal instinct gene regulating zygotropism gene regulating conversion (MZT) just is higher to the requirement of condition of in vitro culture.The embryo is controlled by the maternal effect factor in the ovocyte before female tire conversion, and therefore, activator A effect is not obvious; And conversion back activator A has promoted the secretion of hormone and some factor.Therefore, the present invention is being significantly higher than control group aspect 8/16 cell rate, blastaea rate and the hatching rate that utilize discovery embryo under the activator A effect.And when nutrient solution contained 40ng/mL activator A, embryo's 8/16 cell rate, blastaea rate, hatching rate and ICM/TE were numerically the highest, are respectively 78.0%, 51.6%, 31.8% and 25.2.
2, gene expression doses such as Na/K-ATPase, Glut-1, ActRII, Smad2 significantly improve.
Because Na/K-ATPase is regulating the inside and outside Na of trophocyte
+Concentration is one of essential factor of regulating segmentation cavity formation; Glut-1 raises in normotrophic blastaea, if downward modulation will cause the blastomere number to reduce, even apoptosis; ActRII and Smad2 are the essential molecules of signal conduction in the activator A cell.The present invention has added activator A in nutrient solution, can make gene Na/K-ATPase (0.95 ± 0.3vs.0.42 ± 0.11), Glut-1 (1.33 ± 0.31vs.0.61 ± 0.22) and ActR II (1.53 ± 0.21vs.0.60 ± 0.22), Smad2 (1.37 ± 0.22vs.0.55 ± 0.16) expression level also obviously improve.
Embodiment
A kind of bovine somatic cells clone embryos nutrient solution comprises commercially available bovine somatic cells clone embryos uterine tube nutrient solution (mSOF), and described bovine somatic cells clone embryos uterine tube nutrient solution (mSOF) comprises the CaCl of 2mmol/L
22H
2The KH of the NaCL of O, 107mmol/L, the KCL of 3.2mmol/L, 1.3mmol/L
2PO
4, the activator A of 20～80ng/mL and the water of its surplus, the concentration that facts have proved activator A is 40ng/mL the best.
Wherein activator A is recombinant human activator A or ox activator A, is generally recombinant human activator A, because ox activator A generally is difficult to obtain.
A kind of cultural method that uses bovine somatic cells clone embryos nutrient solution of the present invention, this cultural method be specifically:
1) chooses bovine somatic cells granulosa cell individual layer, 38.5 ℃ of temperature, contain 5%CO
2And under the culture condition of saturated humidity, cultivated 3 days;
2) since the 4th day, adding concentration to commercially available bovine somatic cells clone embryos uterine tube nutrient solution (mSOF) is the activator A of 20～80ng/mL, obtains above-mentioned bovine somatic cells clone embryos nutrient solution, changes liquid every other day once;
3) cultivated 5 days with above-mentioned bovine somatic cells clone embryos nutrient solution again, promptly get bovine somatic cells clone blastaea.Nutrient solution provided by the present invention in use, nutrient solution is 38.5 ℃, 5%CO at culture condition
2, conventional culture condition such as saturated humidity, need change liquid every other day once.
And activator A needed 38.5 ℃ of temperature before commercially available bovine somatic cells clone embryos uterine tube nutrient solution (mSOF) adds, contains 5%CO
2And preheating 20～40 minutes under the condition of saturated humidity, general 30 minutes the bests of preheating.
On the technical system of bovine somatic cells clone embryo two-steps tissue culture method, the present inventor has analyzed the ratio of blastaea rate of formation behind the interpolation activator A, blastomere sum and inner cell mass (ICM) and feeder layer cells (TE), and gene (ActR II and the Smad2) changes of expression level that detects embryo's hatching and implantation genes involved (Na/K-ATPase and Glut-1) and be closely related with activator a-signal path from molecular level.
Blastaea inner cell mass, trophoderm and total cellular score that table 1 was cultivated in the nutrient solution that contains activator A in back 5 days
Different lowercases is represented significant difference on the statistics (P＜0.05) in the same vertical hurdle
Nutrient solution provided by the present invention in use, nutrient solution is 38.5 ℃, 5%CO at culture condition
2, conventional culture condition such as saturated humidity.
And cultivation just may filter out embryo transfer to the acceptor intrauterine on the granulosa cell individual layer after 8 days.Do not contain activator A at nutrient solution in preceding 3 days, and since the 4th day disposable adding activator A, make that activator A is 20～80ng/mL in the nutrient solution, 5 days the nutrient solution in back contains activator A, when the concentration of activator A is 20～80ng/mL, find that embryo's 8/16 cell rate, blastaea rate and hatching rate aspect is significantly higher than control group.
The fetal development situation that table 2 was cultivated in the nutrient solution that contains activator A in back 5 days.
Different lowercases is represented significant difference on the statistics (P＜0.05) in the same vertical hurdle.
When nutrient solution contained 40ng/mL activator A, embryo's 8/16 cell rate, blastaea rate, hatching rate and ICM/TE were numerically the highest, are respectively 78.0%, 51.6%, 31.8%, 25.2.And find that gene Na/K-ATPase (0.95 ± 0.3vs.0.42 ± 0.11), Glut-1 (1.33 ± 0.31vs.0.61 ± 0.22) and ActRII (1.53 ± 0.21vs.0.60 ± 0.22), Smad2 (1.37 ± 0.22vs.0.55 ± 0.16) expression level also obviously improve (notes: the numerical value of vs. front is to have added this expression of gene level behind the activator A, vs. back be this expression of gene level of control group).
Claims (10)
1. a bovine somatic cells clone embryos nutrient solution comprises commercially available bovine somatic cells clone embryos uterine tube nutrient solution (mSOF), and described bovine somatic cells clone embryos uterine tube nutrient solution (mSOF) comprises the CaCl of 1.5～2.5mmol/L
22H
2The KH of the NaCL of O, 100～112mmol/L, the KCL of 2.5～4.0mmol/L, 1.0～1.5mmol/L
2PO
4And the water of its surplus, it is characterized in that: also comprise activator A, the concentration of described activator A is 20～80ng/mL.
2. bovine somatic cells clone embryos nutrient solution according to claim 1 is characterized in that: described bovine somatic cells clone embryos uterine tube nutrient solution (mSOF) comprises the CaCl of 2mmol/L
22H
2The KH of the NaCL of O, 107mmol/L, the KCL of 3.2mmol/L, 1.3mmol/L
2PO
4, the activator A of 20～80ng/mL and the water of its surplus.
3. bovine somatic cells clone embryos nutrient solution according to claim 1 and 2, it is characterized in that: the concentration of activator A is 40ng/mL.
4. bovine somatic cells clone embryos nutrient solution according to claim 3 is characterized in that: described activator A is recombinant human activator A or ox activator A.
5. cultural method that uses the described bovine somatic cells clone embryos of claim 1 nutrient solution is characterized in that this cultural method comprises:
1) chooses bovine somatic cells granulosa cell individual layer, 38.5 ℃ of temperature, contain 5%CO
2And under the culture condition of saturated humidity, cultivated 3 days;
2) since the 4th day, adding concentration to commercially available bovine somatic cells clone embryos uterine tube nutrient solution (mSOF) is the activator A of 20～80ng/mL, obtains the described bovine somatic cells clone embryos of claim 1 nutrient solution.
3) use the described bovine somatic cells clone embryos of claim 1 nutrient solution 5 days again, promptly get the bovine somatic cells clone embryos.
6. according to the described cultural method of claim 5, it is characterized in that: described preceding 3 days cultivation need not changed liquid.
7. according to the described cultural method of claim 5, it is characterized in that: described back 5 days cultivation need be changed liquid once every other day.
8. according to the described cultural method of claim 5, it is characterized in that: need not change liquid in described preceding 3 days, 5 days cultivation then all need be changed liquid once every other day.
9. described according to Claim 8 cultural method is characterized in that: described activator A needed 38.5 ℃ of temperature before commercially available bovine somatic cells clone embryos uterine tube nutrient solution (mSOF) adds, contains 5%CO
2And preheating 20～40 minutes under the condition of saturated humidity.
10. described according to Claim 8 cultural method is characterized in that: described activator A needed 38.5 ℃ of temperature before commercially available bovine somatic cells clone embryos uterine tube nutrient solution (mSOF) adds, contains 5%CO
2And preheating 30 minutes under the condition of saturated humidity.
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| Application Number | Priority Date | Filing Date | Title |
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| CN200910021760A CN101851602A (en) | 2009-03-31 | 2009-03-31 | Solution and method for culturing bovine somatic cell cloned embryos |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719395A (en) * | 2012-06-27 | 2012-10-10 | 中国医学科学院药用植物研究所 | Zebra fish embryo breeding liquid and preparation method thereof |
| CN103305551A (en) * | 2013-06-09 | 2013-09-18 | 西北农林科技大学 | Method for screening donor cell line based on blastocyst gene expression level |
| CN107460208A (en) * | 2017-09-08 | 2017-12-12 | 英科博雅基因科技（天津）有限公司 | Mammalian Somatic Cloning method and the nutrient solution used |
-
2009
- 2009-03-31 CN CN200910021760A patent/CN101851602A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719395A (en) * | 2012-06-27 | 2012-10-10 | 中国医学科学院药用植物研究所 | Zebra fish embryo breeding liquid and preparation method thereof |
| CN103305551A (en) * | 2013-06-09 | 2013-09-18 | 西北农林科技大学 | Method for screening donor cell line based on blastocyst gene expression level |
| CN107460208A (en) * | 2017-09-08 | 2017-12-12 | 英科博雅基因科技（天津）有限公司 | Mammalian Somatic Cloning method and the nutrient solution used |
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Application publication date: 20101006 |