CN106676136B - A method of improving pig nucleus transplantation efficiency - Google Patents
A method of improving pig nucleus transplantation efficiency Download PDFInfo
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- CN106676136B CN106676136B CN201611270696.5A CN201611270696A CN106676136B CN 106676136 B CN106676136 B CN 106676136B CN 201611270696 A CN201611270696 A CN 201611270696A CN 106676136 B CN106676136 B CN 106676136B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
- C12N15/877—Techniques for producing new mammalian cloned embryos
- C12N15/8778—Swine embryos
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
Abstract
The invention discloses a kind of methods for improving pig nucleus transplantation efficiency.The present invention provides a kind of structure embryo mediums, wherein containing 50nM BIX-01294 and 0.5nM chaetocin.The embryo medium can be used to cultivate reconstruct embryo and/or preparation for cultivating the kit of reconstruct embryo.The present invention in PZM-3 culture solution by adding certain density BIX-01294 and chaetocin Combined Treatment early stage clone embryos, the blastocyst rate and blastomere number of clone embryos can be significantly improved, significantly improve the development efficiency of porcine clone embryos, effect than BIX-01294 or chaetocin processing is used alone is more preferable, and the present invention is significant to the whole efficiency for improving pig nucleus transplantation technology.
Description
Technical field
The present invention relates to a kind of methods for improving pig nucleus transplantation efficiency.
Background technique
Somatic cell nuclear transfer technique is born in 1997, succeeds in sheep at first.Its main method is by aobvious
Microoperation and cell-fusion techniques will break up in complete donorcells immigration non-nucleus egg mother cell and constituted reconstructed embryo,
It is cultivated and is transplanted after activation reconstructed embryo, finally obtain complete individuals.Because of the genotype of the individual and donorcells that obtain
It is completely the same, so the technology is also referred to as somatic cell clone technique.Because of its unique technical advantage, somatic cell clone technique is dynamic
Object breeding, disease model building, animals on the brink of extinction protection, therapeutic cloning and pharmaceutical protein production etc. have huge answer
With value.Since first cloned sheep "Dolly" birth in 1997, somatic cell clone technique is in succession in pig, ox, dog, rabbit, cat
Etc. succeeding in a variety of mammals.Although clone technology relative maturity has and a set of includes by vicennial development
The entirety skill such as egg mother cell acquisition, culture, donorcells separation, culture, the activation of ovum, In vitro culture and embryo transfer
Art, but up to the present, the whole efficiency of porcine somatic cell cloning embryos is also very low, about 1% or so.In addition clone pig often occurs
Unsound dysplasia performance, such as lisper, lung's hypoplasia.
Summary of the invention
The object of the present invention is to provide a kind of methods for improving pig nucleus transplantation efficiency.
The present invention provides a kind of reconstruct embryo mediums, wherein containing 50nM BIX-01294 and 0.5nM chaetocin.
The reconstruct embryo medium is basic culture solution with embryo medium.
The embryo medium is PZM-3 culture solution.
The reconstruct embryo medium is made of BIX-01294, chaetocin and the basic culture solution.
The present invention also protects the application of any description above reconstruct embryo medium, is (a1) or (a2):
(a1) culture reconstruct embryo；
(a2) preparation is for cultivating the kit of reconstruct embryo.
The present invention also protects the kit containing any description above reconstruct embryo medium；The purposes of the kit is
Culture reconstruct embryo.
The present invention also protect it is a kind of for cultivate reconstruct embryo kit, the kit includes BIX-01294 and hair
Shell element；The proportion relation of the BIX-01294 and chaetocin is 50nmol BIX-01294:0.5nmol chaetocin.
The kit further includes any description above basic culture solution.
A kind of method that the present invention also protects animal somatic cell nuclear transfer includes the following steps: with any description above weight
Structure embryo medium culture reconstructs embryo.
The method includes two stages；First stage reconstructs embryo medium culture reconstructed embryo with any description above
Tire；Second stage, the culture reconstruct embryo in the culture solution without containing BIX-01294 and chaetocin.
The first stage is 14-16h, specially 16h.
The culture solution without containing BIX-01294 and chaetocin concretely any description above basic culture solution.
The preparation method of the reconstruct embryo is concretely: donorcells are injected into the egg mother cell after stoning
In perivitelline, electric shock merges and activates reconstruct embryo.
It is described to shock by electricity the condition merged concretely: the electric pulse of 150v/mm, 100 μ s, 2DC.
The preparation method of " egg mother cell after stoning " is concretely: female thin using blind suction method removal animal mature egg
Born of the same parents' first polar body and nucleus.
The donorcells concretely foetal animal fibroblast.
The fibroblastic preparation method of foetal animal specifically may include following steps (b1) and step (b2):
(b1) in vitro fetus is taken, cuts off except head, four limbs and internal organ, remainder is shredded and is transferred in culture dish, In
37 DEG C, saturated humidity, 5%CO2Incubator in cultivate 4-6 hours；
(b2) it after completing step (b1), is added in Xiang Suoshu culture dish containing 15% (volumn concentration) fetal calf serum
DMEM culture solution is cultivated and is passed on, and takes the fetus for the contact inhibition for being passaged to 3-5 generation (concretely the 3rd generation) at fiber
Cell.
The preparation method of the animal mature oocyte specifically may include following steps: (c1)-(c3):
(c1) in vitro animal ovary is taken, the liquor folliculi in ovarian follicle is extracted, selects and be enclosed with 3-5 layers of cumulus cell
Cumulus oocyte complex (COCs)；
(c2) cumulus oocyte complex (COCs) that step (c1) obtains is transferred in IVM liquid, in 39 DEG C, saturation
Humidity, 5%CO242h is cultivated in incubator.
(c3) it after completing step (c2), with cumulus cell is removed after 0.1% hyaluronic acid enzymic digestion, selects with the first pole
The mature oocyte of body and cytoplasm even compact.
The IVM liquid is made of solvent and solute, the concentration in the solute and IVM liquid are as follows: the solute and its in IVM
Concentration in liquid are as follows: 199 culture medium 0.987g/100mL, liquor folliculi 10mL/100mL, 1 μ g/100mL of epidermal growth factor, promote
Follicular hormone 50mg/100mL, metakentrin 50mg/100mL, 7 μ g/100mL of cysteine；The solvent is pure water.
Any description above animal concretely mammal, more specifically can be pig.
The present invention in PZM-3 culture solution by adding certain density BIX-01294 and chaetocin Combined Treatment early stage
Clone embryos can significantly improve the blastocyst rate and blastomere number of clone embryos, significantly improve the development efficiency of porcine clone embryos,
Effect than BIX-01294 or chaetocin processing is used alone is more preferable, and the present invention is to raising pig nucleus transplantation technology
Whole efficiency is significant.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with and repeats to test more than three times, as a result
It is averaged.
199 culture mediums: Gibco, article No.: 31100-035.
Epidermal growth factor: Promega, article No.: G5021.
Follicle-stimulating hormone (FSH): Sigma, article No.: F8174.
Metakentrin: Sigma, article No.: L5269.
IVM liquid (In-vitro maturation liquid) is made of solvent and solute, the solute and its concentration in IVM liquid are as follows:
199 culture medium 0.987g/100mL, liquor folliculi 10mL/100mL, 1 μ g/100mL of epidermal growth factor, follicle-stimulating hormone (FSH) 50mg/
100mL, metakentrin 50mg/100mL, 7 μ g/100mL of cysteine；The solvent is pure water.
BIX-01294:Sigma, article No.: B9311；The molecular formula of BIX-01294 is C28H38N6O23HCl, No. CAS:
1392399-03-9, structural formula are as follows:
Chaetocin: Sigma, article No.: C9492；The molecular formula of chaetocin is C30H28N6O684, No. CAS: 28097-03-2,
Structural formula is as follows:
DPBS buffer: Sigma, article No.: D5652.
Embodiment 1, pig nucleus transplantation
One, the In-vitro maturation of porcine oocytes
Pig ovary is taken from slaughterhouse, with the normal saline flushing three containing 100U/mL penicillin and 100U/mL streptomysin
Time, then extracting diameter with the disposable syringe of 10mL specification and No. 18 syringe needles is the liquor folliculi in the ovarian follicle of 3-6mm, therefrom
Pick out the cumulus oocyte complex (COCs) for being enclosed with 3-5 layers of cumulus cell.COCs use containing 100U/mL penicillin and
Twice of the normal saline flushing of 100U/mL streptomysin is then transferred into IVM liquid, in 39 DEG C, saturated humidity, 5%CO2Culture
42h is cultivated in case.
Two, the separation and culture of porcine fetus fibroblasts
Porcine fetus fibroblasts are successively prepared in accordance with the following steps:
1, from gestation 35 days farrowing sow uterus in take out fetus, with contain 100U/mL penicillin and 100U/mL chain
The DPBS buffer of mycin rinses.
2, the fetus for taking step 1 to obtain removes head, four limbs and internal organ with eye scissors in superclean bench, by remainder
Shredded and be transferred in 100mm culture dish with the eye scissors to sterilize after point being rinsed with DPBS buffer, 37 DEG C, saturated humidity,
5%CO2Incubator in cultivate 4-6 hours.
3, after completing step 2, the DMEM for containing 15% (volumn concentration) fetal calf serum is added in Xiang Suoshu culture dish
Culture solution, in 37 DEG C, saturated humidity, 5%CO2Incubator in culture to cell confluency degree be 90% when secondary culture.Passage
The culture solution used is cultivated as the DMEM culture solution containing 15% (volumn concentration) fetal calf serum.
4, subsequent experimental is carried out with the fetal fibroblast for the contact inhibition for being passaged to for the 3rd generation.
Three, body-cell neucleus transplanting
1, the COCs for taking into step 1, with cumulus cell is removed after 0.1% hyaluronic acid enzymic digestion, then in IVM liquid
Middle cleaning three times, selects the mature oocyte with first polar body and cytoplasm even compact.
2, " mature oocyte with first polar body and cytoplasm even compact " that step 1 obtains is taken, the first pole is removed
Body and surrounding cytoplasm, being then injected into a donor cell, (fetus that donor cell i.e. step 2 obtains is at fiber finer
Born of the same parents), and be located in perivitelline.
3, complete step 2 after, the egg mother cell for injecting donor cell is moved on in integration slot, using 150v/mm, 100 μ s,
The electric pulse electric shock of 2DC merges and activates reconstruct embryo.
4, the reconstruct embryo for obtaining step 3 according to carry out following grouping culture (condition of culture are as follows: 39 DEG C, saturated humidity,
5%CO2):
Control group: reconstruct embryo is put into PZM-3 culture solution and is cultivated 7 days.
Experimental group A: reconstruct embryo is put into the PZM-3 culture solution containing 50nMBIX-01294 and cultivates 16h, Zhi Houzhuan
It moves in PZM-3 culture solution and cultivates 7 days.
Experimental group B: reconstruct embryo is put into the PZM-3 culture solution containing 0.5nM chaetocin and cultivates 16h, is shifted later
It is cultivated 7 days into PZM-3 culture solution.
Experimental group C: reconstruct embryo is put into the PZM-3 culture solution containing 50nMBIX-01294 and 0.5nM chaetocin and is trained
16h is supported, is transferred in PZM-3 culture solution and cultivates 7 days later.
Experimental group D: reconstruct embryo is put into the PZM-3 culture solution containing 50nMBIX-01294 and 1.0nM chaetocin and is trained
16h is supported, is transferred in PZM-3 culture solution and cultivates 7 days later.
3, spilting of an egg number and blastaea number are observed and recorded at the 24th hour of culture and the 7th day.The results are shown in Table 1.
The different group spilting of an egg numbers of table 1 and blastaea number statistical result
Note: 4 repetition test datas of statistical analysis calculate its average value ± standard error.Different small letters in same row
Female subscript represents significant difference (P < 0.05)
The result shows that the porcine clone embryos handled simultaneously using 50nMBIX-01294 and 0.5nM chaetocin, capsule at the 7th day
Embryo rate is significantly higher than control group, BIX-01294 processing is used alone and the embryo of chaetocin processing is used alone.
Claims (3)
1. a kind of reconstruct embryo medium, is made of BIX-01294, chaetocin and basic culture solution；
The concentration of the BIX-01294 is 50nM；
The concentration of the chaetocin is 0.5nM；
The basic culture solution is PZM-3 culture solution.
2. reconstruct embryo medium described in claim 1 is in preparation for cultivating the application in the kit for reconstructing embryo.
3. containing the kit for reconstructing embryo medium described in claim 1；The purposes of the kit is culture reconstruct embryo.
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| CN106676136B true CN106676136B (en) | 2019-11-29 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104278008A (en) * | 2013-07-12 | 2015-01-14 | 北京大学科技开发部 | Method, kit and applications of preparing pluripotent stem cells through small-molecule compound treatment |
| CN104673741A (en) * | 2015-02-04 | 2015-06-03 | 广东省中医院 | High-efficiency non-integrated human iPSC induction platform |
| WO2016044271A2 (en) * | 2014-09-15 | 2016-03-24 | Children's Medical Center Corporation | Methods and compositions to increase somatic cell nuclear transfer (scnt) efficiency by removing histone h3-lysine trimethylation |
| CN105543230A (en) * | 2016-03-02 | 2016-05-04 | 华南农业大学 | Method for improving pig cloning efficiency on basis of inhibition of H3K9me3 methylation |
-
2016
- 2016-12-30 CN CN201611270696.5A patent/CN106676136B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104278008A (en) * | 2013-07-12 | 2015-01-14 | 北京大学科技开发部 | Method, kit and applications of preparing pluripotent stem cells through small-molecule compound treatment |
| WO2016044271A2 (en) * | 2014-09-15 | 2016-03-24 | Children's Medical Center Corporation | Methods and compositions to increase somatic cell nuclear transfer (scnt) efficiency by removing histone h3-lysine trimethylation |
| CN104673741A (en) * | 2015-02-04 | 2015-06-03 | 广东省中医院 | High-efficiency non-integrated human iPSC induction platform |
| CN105543230A (en) * | 2016-03-02 | 2016-05-04 | 华南农业大学 | Method for improving pig cloning efficiency on basis of inhibition of H3K9me3 methylation |
Non-Patent Citations (2)
| Title |
|---|
| BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei;Jiaojiao Huang等;《Reproduction》;20160131;第151卷(第1期);摘要，第41页左栏"Postactivation and embryo culture"部分，第43页右栏第2段以及表1以及表2 * |
| 组蛋白甲基转移酶在造血干细胞调控和白血病发生中的作用;王晓娟等;《中华血液学杂志》;20120930;第33卷(第9期);第481-484页 * |
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Effective date of registration: 20210511 Address after: Building A8, phase I, Nanning ASEAN enterprise headquarters port, No.10, Xinji Road, high tech Zone, Nanning, Guangxi 530000 Patentee after: Nanning zhuangbo Biotechnology Co.,Ltd. Address before: 100193 Beijing Institute of animal husbandry and veterinary medicine, Chinese Academy of Agricultural Sciences, 2 West Old Summer Palace Road, Beijing, Haidian District Patentee before: Beijing Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences |