CN106591374B - A method of improving pig nucleus transplantation embryonic development efficiency - Google Patents
A method of improving pig nucleus transplantation embryonic development efficiency Download PDFInfo
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- CN106591374B CN106591374B CN201611270683.8A CN201611270683A CN106591374B CN 106591374 B CN106591374 B CN 106591374B CN 201611270683 A CN201611270683 A CN 201611270683A CN 106591374 B CN106591374 B CN 106591374B
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- 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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- 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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- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/74—Undefined extracts from fungi, e.g. yeasts
Abstract
The invention discloses a kind of methods for improving pig nucleus transplantation embryonic development efficiency.The present invention provides the applications of chaetocin, are used for the donor cell culture solution of animal somatic cell nuclear transfer at least one of following (a1)-(a5): (a1) preparation；(a2) preparation is used for the reconstruct embryo medium of animal somatic cell nuclear transfer；(a3) culture is used for the donor cell of animal somatic cell nuclear transfer；(a4) culture is used for the reconstruct embryo of animal somatic cell nuclear transfer；(a5) promote the reconstruct embryogenesis blastaea in animal somatic cell nuclear transfer.The present invention by using small-molecule drug chaetocin in body-cell neucleus transplanting donor cell or reconstruct embryo handle, promote the generation of body-cell neucleus transplanting reprogramming, the blastocyst rate and blastomere number of clone embryos are significantly improved, the final whole efficiency for improving pig nucleus transplantation technology.The present invention is significant to the whole efficiency for improving pig nucleus transplantation technology.
Description
Technical field
The present invention relates to embryonic development and Animal Biotechnology fields, and in particular to a kind of raising pig nucleus transplantation
The method of embryonic development 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 donor cell 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 donor cell 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, donor cell 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 embryonic development efficiency.
The present invention provides the applications of chaetocin, are at least one of following (a1)-(a5):
(a1) preparation is used for the donor cell culture solution of animal somatic cell nuclear transfer；
(a2) preparation is used for the reconstruct embryo medium of animal somatic cell nuclear transfer；
(a3) culture is used for the donor cell of animal somatic cell nuclear transfer；
(a4) culture is used for the reconstruct embryo of animal somatic cell nuclear transfer；
(a5) promote the reconstruct embryogenesis blastaea in animal somatic cell nuclear transfer.
The present invention also protects a kind of donor cell culture solution for animal somatic cell nuclear transfer, wherein containing 10nM maos of shells
Element.
The donor cell culture solution includes chaetocin and basal cell culture solution.
The basal cell culture solution concretely DMEM culture solution.
The donor cell culture solution further includes 15% (percent by volume) fetal calf serum.
The donor cell culture solution further includes 100U/mL ampicillin and 100U/mL streptomycin sulphate.
The present invention also protects a kind of reconstruct embryo medium for animal somatic cell nuclear transfer, wherein containing 0.5nM maos
Shell element.
The reconstruct embryo medium includes chaetocin and basic embryo medium.
The basis embryo medium concretely PZM-3 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
Donor cell culture solution culture donor cell.
The incubation time is concretely for 24 hours.
The method also includes the donor cells for taking culture to terminate to be used to prepare reconstruct embryo.
The preparation method of the reconstruct embryo is concretely: the donor cell that one is obtained by the method culture is infused
In the perivitelline for entering the egg mother cell to stoning, electric shock merges and activates reconstruct embryo.
A kind of method that the present invention also protects animal somatic cell nuclear transfer includes the following steps: with any description above
It reconstructs embryo medium culture and reconstructs embryo.
The method includes the cultures in two stages；First stage is trained with the reconstruct embryo medium of any description above
Support reconstruct embryo；Second stage, the culture reconstruct embryo in the culture solution without containing chaetocin.
It is described not contain the concretely any description above basis embryo medium of the culture solution without containing chaetocin.
The first stage is 14-16h, specially 16h.
The preparation method of the reconstruct embryo is concretely: donor cell is injected into the egg mother cell after stoning
In perivitelline, electric shock merges and activates reconstruct embryo.
Any description above shocks by electricity the condition merged concretely: the electric pulse of 150v/mm, 100 μ s, 2DC.
The preparation method of any description above " egg mother cell after stoning " is concretely: using blind suction method removal animal at
Ripe egg mother cell first polar body and nucleus.
Any description above 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,
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 any description above 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.
Chaetocin (chaetocin) is a kind of natural bioactive substances for being isolated from Chaetomium (Chaetomium) fungi, it is right
The growth of tumour cell has certain inhibiting effect, therefore by as a kind of potential tumor therapeutic agent.Chaetocin is in body
Cell reprograms field and never applies, and does not have chaetocin for promoting the report in terms of embryonic development at present.
The present invention is by the donor cell in body-cell neucleus transplanting or reconstructing embryo using small-molecule drug chaetocin
It is handled, thus the generation for promoting body-cell neucleus transplanting to reprogram, and then the blastocyst rate of clone embryos can be significantly improved
With blastomere number, the final whole efficiency for improving pig nucleus transplantation technology.The present invention is to raising pig nucleus transplantation
The whole efficiency of technology is significant.
Detailed description of the invention
Fig. 1 is activity influence of the chaetocin to porcine fetus fibroblasts.
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 three repeated experiments, as a result makes even
Mean value.
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.
Chaetocin: Sigma, article No.: C9492；The molecular formula of chaetocin is C30H28N6O6S4, No. CAS: 28097-03-2,
Structural formula is as follows:
DPBS buffer: Sigma, article No.: D5652.
The In-vitro maturation of embodiment 1, 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.
The separation and culture of embodiment 2, 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 porcine fetus fibroblasts for being passaged to for the 3rd generation.
The influence of embodiment 3, chaetocin to porcine fetus fibroblasts
1, the porcine fetus fibroblasts of logarithmic growth phase prepared by embodiment 2 are seeded to 96 orifice plates (every hole 5 × 104
A cell), using 37 DEG C of stationary cultures of DMEM culture solution containing 15% (volumn concentration) fetal calf serum until cell pastes
Wall.
2, after completing step 2,96 orifice plate is taken, following packet transaction is carried out:
Medicine group 1: inhaling and abandon culture supernatant, and the DMEM culture solution (15% fetal calf serum+100U/ of the chaetocin containing 20nM is added
ML ampicillin+100U/mL streptomycin sulphate), 37 DEG C stand 24 hours；
Medicine group 2: inhaling and abandon culture supernatant, and the DMEM culture solution (15% fetal calf serum+100U/ of the chaetocin containing 40nM is added
ML ampicillin+100U/mL streptomycin sulphate), 37 DEG C stand 24 hours；
Medicine group 3: inhaling and abandon culture supernatant, and the DMEM culture solution (15% fetal calf serum+100U/ of the chaetocin containing 50nM is added
ML ampicillin+100U/mL streptomycin sulphate), 37 DEG C stand 24 hours；
Medicine group 4: inhaling and abandon culture supernatant, and the DMEM culture solution (15% fetal calf serum+100U/ of the chaetocin containing 100nM is added
ML ampicillin+100U/mL streptomycin sulphate), 37 DEG C stand 24 hours；
Control group: inhaling and abandon culture supernatant, is added DMEM culture solution (15% fetal calf serum+0.1% is dual anti-).
Respectively after treatment 12h and for 24 hours when detect cell activity.
As a result as shown in Figure 1.The result shows that higher than 40nM concentration chaetocin to the toxicity of porcine fetus fibroblasts compared with
Greatly.40nM chaetocin processing porcine fetus fibroblasts for 24 hours after, cell activity only residue 50% or so.
Embodiment 4, pig nucleus transplantation
One, chaetocin handles porcine fetus fibroblasts
1, the porcine fetus fibroblasts that embodiment 2 obtains are seeded in 6 hole culture dishes (every ware 1 × 106A cell),
Using 37 DEG C of stationary cultures of DMEM culture solution containing 15% (volumn concentration) fetal calf serum until cell is long to 90% remittance
It is right.
2, after completing step 2, the culture dish is taken, following packet transaction is carried out:
Control group: inhaling and abandon culture supernatant, and the DMEM culture solution (15% fetal calf serum+100U/mL of the chaetocin containing 0nM is added
Ampicillin+100U/mL streptomycin sulphate), 37 DEG C stand 24 hours；
Medicine group 1: inhaling and abandon culture supernatant, and the DMEM culture solution (15% fetal calf serum+100U/ of the chaetocin containing 10nM is added
ML ampicillin+100U/mL streptomycin sulphate), 37 DEG C stand 24 hours；
Medicine group 2: inhaling and abandon culture supernatant, and the DMEM culture solution (15% fetal calf serum+100U/ of the chaetocin containing 20nM is added
ML ampicillin+100U/mL streptomycin sulphate), 37 DEG C stand 24 hours；
Medicine group 3: inhaling and abandon culture supernatant, and the DMEM culture solution (15% fetal calf serum+100U/ of the chaetocin containing 40nM is added
ML ampicillin+100U/mL streptomycin sulphate), 37 DEG C stand 24 hours.
3, the COCs that Example 1 obtains, 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.
4, " mature oocyte with first polar body and cytoplasm even compact " that step 3 obtains is taken, the first pole is removed
Body and surrounding cytoplasm, being then injected into a donor cell, (donor cell i.e. step 2 is obtained by packet transaction culture
Porcine fetus fibroblasts), and be located in perivitelline.
5, complete step 4 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.
6, the reconstruct embryo that step 3 obtains is put into PZM-3 culture solution and cultivates 7 days (condition of culture are as follows: 39 DEG C, saturation
Humidity, 5%CO2)。
7, spilting of an egg number is observed and recorded within the 48th hour in culture, the 7th day when observes and records spilting of an egg number blastaea number.As a result such as table
Shown in 1.
The different group spilting of an egg numbers of table 1 and blastaea number statistical result
Note: 3 repetition test datas of statistical analysis calculate its average value ± standard error.All percentage datas are by anti-
T- inspection is carried out after sine conversion, the different lowercase subscripts in same row represent significant difference (P < 0.05).
The result shows that handling porcine fetus fibroblasts (donor cell) using 10nM chaetocin, blastocyst rate is aobvious at the 7th day
It writes and is higher than control group and other medicines group.
Two, chaetocin processing reconstruct embryo
1, the COCs that Example 1 is cultivated, 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. embodiment 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 0.5nM chaetocin and cultivates 16h, is shifted later
It is cultivated 7 days into PZM-3 culture solution.
Experimental group B: reconstruct embryo is put into the PZM-3 culture solution containing 1.0nM chaetocin and cultivates 16h, is shifted later
It is cultivated 7 days into PZM-3 culture solution.
3, spilting of an egg number is observed and recorded within the 48th hour in culture, the 7th day when observes and records spilting of an egg number blastaea number.As a result such as table
Shown in 2.
The different group spilting of an egg numbers of table 2 and blastaea number statistical result
Note: 4 repetition test datas of statistical analysis calculate its average value ± standard error.All percentage datas are by anti-
T- inspection is carried out after sine conversion, the different lowercase subscripts in same row represent significant difference (P < 0.05).
The result shows that handling processing reconstruct embryo using 0.5nM chaetocin, blastocyst rate is significantly higher than control group at the 7th day
With 1.0nM chaetocin group.
Claims (2)
1. a kind of method of animal somatic cell nuclear transfer includes the following steps: with donor cell culture solution culture donor cell；Institute
It states and contains 10nM chaetocin in donor cell culture solution；The donor cell culture solution includes chaetocin and basal cell culture
Liquid；The basal cell culture solution is DMEM culture solution；
The donor cell is porcine fetus fibroblasts；
The method is non-disease treatment method.
2. a kind of method of animal somatic cell nuclear transfer, it is characterised in that: the method includes the cultures in two stages；First rank
Section reconstructs embryo with reconstruct embryo medium culture；Second stage cultivates reconstructed embryo in the culture solution without containing chaetocin
Tire；Contain 0.5nM chaetocin in the reconstruct embryo medium；
The first stage is 16h；
It is described reconstruct embryo's the preparation method comprises the following steps: by a donor cell be injected into stoning after egg mother cell perivitelline in,
Electric shock merges and activates reconstruct embryo；
The condition of the electric shock fusion are as follows: the electric pulse of 150v/mm, 100 μ s, 2DC；
" egg mother cell after stoning " the preparation method comprises the following steps: using blind suction method removal pig mature oocyte first polar body and
Nucleus；
The preparation method of the pig mature oocyte includes the following steps: (c1)-(c3):
(c1) in vitro pig ovary is taken, the liquor folliculi in ovarian follicle is extracted, selects the ovarian cumulus ovum for being enclosed with 3-5 layers of cumulus cell
Mother cell complex (COCs)；
(c2) cumulus oocyte complex (COCs) that step (c1) obtains is transferred in IVM liquid, 39 DEG C, saturation it is wet
Degree, 5% CO242h is cultivated in incubator；
(c3) after completing step (c2), with cumulus cell is removed after 0.1% hyaluronic acid enzymic digestion, select with first polar body and
The mature oocyte of cytoplasm even compact；
The IVM 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；
The reconstruct embryo medium includes chaetocin and basic embryo medium；The basis embryo medium is PZM-3 training
Nutrient solution；
The method is non-disease treatment method.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103550222A (en) * | 2013-11-05 | 2014-02-05 | 南京医科大学 | Applications of chaetocin in preparing medicament for preventing and treating diabetes |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103550222A (en) * | 2013-11-05 | 2014-02-05 | 南京医科大学 | Applications of chaetocin in preparing medicament for preventing and treating diabetes |
| 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 |
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