CN104726397A - Breeding method of denuded oocyte mice - Google Patents
Breeding method of denuded oocyte mice Download PDFInfo
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- CN104726397A CN104726397A CN201510113691.0A CN201510113691A CN104726397A CN 104726397 A CN104726397 A CN 104726397A CN 201510113691 A CN201510113691 A CN 201510113691A CN 104726397 A CN104726397 A CN 104726397A
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Abstract
The invention discloses a breeding method of denuded oocyte mice. The breeding method comprises two steps of ectogenesis of mice denuded oocytes and mice denuded oocyte embryos. According to the breeding method, combined application is carrying out by simulating a natural development mode in a maturation process of oocytes by virtue of Forskolin and PD166285, the mice denuded oocyte embryos can mature under complete in vitro culture conditions by virtue of the combined application of Forskolin and PD166285 without an exogenous follicular development hormone, and the mature denuded oocytes can be subjected to in vitro fertilization to give birth to new individual mice, the birthing rate of the new denuded oocyte mice born form the mice denuded oocytes can reach 11.11%, and the newly born denuded oocyte mice can develop normally.
Description
Technical field
The present invention relates to a kind of ova nuda mouse production method.
Background technology
Ovary is the source region that ovum produces, and ovum is the important sexual cell that nature new life is born.In its natural state, entovarial ovum is wrapped up by the cumulus cell that surrounding is a large amount of, and cumulus cell plays important impact for the ripe quality of ovum and developmental potentiality.In modern society, along with in the face of the competition of various survival pressure and the decline of human physique, there is apoptosis and decline in the cumulus cell around ovum, forms a large amount of ova nuda in ovary, this namely known early ageing.Under normal circumstances, the ova nuda in these ovaries can not normal mature, can not combine the living individual and offspring that are born and make new advances with sperm.In modern times infertile crowd and animal individual, such colony occupies higher ratio.Therefore, healthy reproduction becomes the major issue that the mankind face day by day.Meanwhile, the livestock product of human demand's high yield and high quality in Modern Animal Husbandry, these need the Animal Group of high yield and high quality, and the cultivation of outstanding Animal Group is particularly important.Body somatocyte can induced synthesis stem cell in vitro, the associated biomolecule breeding techniques such as stem cell have developed rapidly and maturation, stem cell can form ova nuda by induced development in vitro, therefore, can the be born individuality that makes new advances of the ova nuda of somatic sources will provide Important Platform and guarantee for the modern development of cattle breeding.
How mouse, as the important animal model of research reproductive development, makes mouse denuded oocyte reach maturity under culture condition completely in vitro, and ripe ova nuda can carry out in vitro fertilization and be born and normal newborn individual mice, is a problem being worth research.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of ova nuda mouse production method.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind of ova nuda mouse production method, comprises the following steps:
(1) ectogenesis of mouse denuded oocyte
Get mouse ovarian, puncture ovarian follicle on ovary with syringe needle, in ovarian follicle, ovocyte is discharged in ovocyte operation liquid; After selecting the external washing of ova nuda, put into the oocyte maturation nutrient solution containing 50 μMs of Forskolin, ova nuda is transferred in the oocyte maturation nutrient solution containing 2.5 μMs of PD166285 and continues vitro culture 16 h cultivate 3 h in the oocyte maturation nutrient solution containing 50 μMs of Forskolin after;
(2) ectogenesis of mouse denuded oocyte embryo
Post-mature of learning from else's experience cultivates the ova nuda of discharging first polar body, at HTF by seminal fluid and capacitated sperm is external jointly hatches cultivation 6 h, select the zygote discharged second polar body or form female-male pronucleus, cultivate in G-1 PLUS nutrient solution, it is ripe that ova nuda is in vitro fertilization when being developed to 2-cell stage, transplanting is developed to the mouse denuded oocyte embryo of 2-cell stage in replace-conceive Mouse Oviductal, raises replace-conceive mouse and within 20 days, obtains ova nuda mouse afterwards in embryo transfer.
As preferably, ovocyte operation liquid is the TCM199 containing HEPES and 10% FBS.
Preferred as another, oocyte maturation nutrient solution is SAGE
?in-Vitro Maturation Media.
The invention has the beneficial effects as follows:
In vitro under envrionment conditions, utilize Forskolin and PD166285 to simulate natural development models in Oocyte Maturation Process and carry out combined utilization, do not having under external source follicular development functions of hormones, Forskolin and PD166285 combined utilization can make mouse denuded oocyte reach maturity under culture condition completely in vitro, ripe ova nuda can carry out the in vitro fertilization and individual mice made new advances that is born, the mouse denuded oocyte farrowing rate newborn mice that is born can reach 11.11%, and newborn ova nuda mice develop is normal.
Embodiment
A kind of ova nuda mouse production method, concrete steps are as follows:
(1) ectogenesis of mouse denuded oocyte
Get mouse ovarian, puncture ovarian follicle on ovary with syringe needle, in ovarian follicle, ovocyte is discharged in ovocyte operation liquid, and ovocyte operation liquid is the TCM199 containing HEPES and 10% FBS; After selecting the external washing of ova nuda, put into the oocyte maturation nutrient solution SAGE containing 50 μMs of Forskolin
?in In-Vitro Maturation Media, ova nuda is transferred to the oocyte maturation nutrient solution SAGE containing 2.5 μMs of PD166285 cultivate 3 h in the oocyte maturation nutrient solution containing 50 μMs of Forskolin after
?vitro culture 16 h is continued in In-Vitro Maturation Media;
(2) ectogenesis of mouse denuded oocyte embryo
Post-mature of learning from else's experience cultivates the ova nuda of discharging first polar body, at HTF by seminal fluid and capacitated sperm is external jointly hatches cultivation 6 h, select the zygote discharged second polar body or form female-male pronucleus, cultivate in G-1 PLUS nutrient solution, it is ripe that ova nuda is in vitro fertilization when being developed to 2-cell stage, transplanting is developed to the mouse denuded oocyte embryo of 2-cell stage in replace-conceive Mouse Oviductal, raises replace-conceive mouse and within 20 days, obtains ova nuda mouse afterwards in embryo transfer.
Above-described embodiment of the present invention, does not form limiting the scope of the present invention.Any amendment done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within claims of the present invention.
Claims (3)
1. an ova nuda mouse production method, is characterized in that comprising the following steps:
(1) ectogenesis of mouse denuded oocyte
Get mouse ovarian, puncture ovarian follicle on ovary with syringe needle, in ovarian follicle, ovocyte is discharged in ovocyte operation liquid; After selecting the external washing of ova nuda, put into the oocyte maturation nutrient solution containing 50 μMs of Forskolin, ova nuda is transferred in the oocyte maturation nutrient solution containing 2.5 μMs of PD166285 and continues vitro culture 16 h cultivate 3 h in the oocyte maturation nutrient solution containing 50 μMs of Forskolin after;
(2) ectogenesis of mouse denuded oocyte embryo
Post-mature of learning from else's experience cultivates the ova nuda of discharging first polar body, at HTF by seminal fluid and capacitated sperm is external jointly hatches cultivation 6 h, select the zygote discharged second polar body or form female-male pronucleus, cultivate in G-1 PLUS nutrient solution, it is ripe that ova nuda is in vitro fertilization when being developed to 2-cell stage, transplanting is developed to the mouse denuded oocyte embryo of 2-cell stage in replace-conceive Mouse Oviductal, raises replace-conceive mouse and within 20 days, obtains ova nuda mouse afterwards in embryo transfer.
2. ova nuda mouse production method according to claim 1, is characterized in that, described ovocyte operation liquid is the TCM199 containing HEPES and 10% FBS.
3. ova nuda mouse production method according to claim 1, is characterized in that, described oocyte maturation nutrient solution is SAGE In-Vitro Maturation Media.
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| CN201510113691.0A CN104726397A (en) | 2015-03-16 | 2015-03-16 | Breeding method of denuded oocyte mice |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024087350A1 (en) * | 2022-10-27 | 2024-05-02 | 南京医科大学 | Method for constructing short-telomere mouse model |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101560494A (en) * | 2009-05-31 | 2009-10-21 | 山东农业大学 | Mouse denuded oocyte in vitro maturation technology |
| CN103898048A (en) * | 2014-03-27 | 2014-07-02 | 安徽农业大学 | In vitro maturation culture method of denuded oocytes of mice |
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2015
- 2015-03-16 CN CN201510113691.0A patent/CN104726397A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101560494A (en) * | 2009-05-31 | 2009-10-21 | 山东农业大学 | Mouse denuded oocyte in vitro maturation technology |
| CN103898048A (en) * | 2014-03-27 | 2014-07-02 | 安徽农业大学 | In vitro maturation culture method of denuded oocytes of mice |
Non-Patent Citations (1)
| Title |
|---|
| CORTVRINDT R G等: "Timed analysis of the nuclear maturation of oocytes in early preantral mouse follicle culture supplemented with recombmant gonadotropin", 《FERTILITY AND STERILITY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024087350A1 (en) * | 2022-10-27 | 2024-05-02 | 南京医科大学 | Method for constructing short-telomere mouse model |
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