CN107937444A - The method of somatic cell clone dog - Google Patents
The method of somatic cell clone dog Download PDFInfo
- Publication number
- CN107937444A CN107937444A CN201710614323.3A CN201710614323A CN107937444A CN 107937444 A CN107937444 A CN 107937444A CN 201710614323 A CN201710614323 A CN 201710614323A CN 107937444 A CN107937444 A CN 107937444A
- Authority
- CN
- China
- Prior art keywords
- cell
- bitch
- acceptor
- ovum
- clone
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 41
- 210000001082 somatic cell Anatomy 0.000 title claims abstract description 24
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 39
- 230000004927 fusion Effects 0.000 claims abstract description 34
- 239000007788 liquid Substances 0.000 claims abstract description 28
- 210000004027 cell Anatomy 0.000 claims description 48
- 210000000287 oocyte Anatomy 0.000 claims description 41
- 108010000912 Egg Proteins Proteins 0.000 claims description 34
- 102000002322 Egg Proteins Human genes 0.000 claims description 34
- 210000004681 ovum Anatomy 0.000 claims description 34
- 230000007159 enucleation Effects 0.000 claims description 29
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 claims description 22
- 210000000130 stem cell Anatomy 0.000 claims description 20
- 238000002054 transplantation Methods 0.000 claims description 16
- 230000003204 osmotic effect Effects 0.000 claims description 15
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- 210000000805 cytoplasm Anatomy 0.000 claims description 14
- 229960003387 progesterone Drugs 0.000 claims description 11
- 239000000186 progesterone Substances 0.000 claims description 11
- 210000001519 tissue Anatomy 0.000 claims description 11
- 230000016087 ovulation Effects 0.000 claims description 10
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 8
- 210000004369 blood Anatomy 0.000 claims description 8
- 239000008280 blood Substances 0.000 claims description 8
- 210000002257 embryonic structure Anatomy 0.000 claims description 8
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 7
- 229930195725 Mannitol Natural products 0.000 claims description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- 235000010355 mannitol Nutrition 0.000 claims description 7
- 239000000594 mannitol Substances 0.000 claims description 7
- 210000003101 oviduct Anatomy 0.000 claims description 7
- 210000002950 fibroblast Anatomy 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 210000001672 ovary Anatomy 0.000 claims description 6
- 210000003491 skin Anatomy 0.000 claims description 6
- 230000002146 bilateral effect Effects 0.000 claims description 5
- 238000012545 processing Methods 0.000 claims description 5
- 210000004204 blood vessel Anatomy 0.000 claims description 3
- 210000002889 endothelial cell Anatomy 0.000 claims description 3
- 210000003038 endothelium Anatomy 0.000 claims description 3
- 210000002919 epithelial cell Anatomy 0.000 claims description 3
- 230000001605 fetal effect Effects 0.000 claims description 3
- 210000003754 fetus Anatomy 0.000 claims description 3
- 230000000762 glandular Effects 0.000 claims description 3
- 210000002149 gonad Anatomy 0.000 claims description 3
- 210000002510 keratinocyte Anatomy 0.000 claims description 3
- 210000003205 muscle Anatomy 0.000 claims description 3
- 210000000663 muscle cell Anatomy 0.000 claims description 3
- 210000000056 organ Anatomy 0.000 claims description 3
- 210000000963 osteoblast Anatomy 0.000 claims description 3
- 210000004927 skin cell Anatomy 0.000 claims description 3
- -1 urine Substances 0.000 claims description 3
- 210000002700 urine Anatomy 0.000 claims description 3
- 230000002611 ovarian Effects 0.000 claims description 2
- 210000005075 mammary gland Anatomy 0.000 claims 2
- 230000035800 maturation Effects 0.000 claims 2
- 210000002569 neuron Anatomy 0.000 claims 2
- 210000000981 epithelium Anatomy 0.000 claims 1
- 238000012546 transfer Methods 0.000 abstract description 19
- 238000005516 engineering process Methods 0.000 abstract description 11
- 241000282472 Canis lupus familiaris Species 0.000 description 73
- 241001465754 Metazoa Species 0.000 description 11
- 239000007924 injection Substances 0.000 description 11
- 238000002347 injection Methods 0.000 description 11
- 230000012173 estrus Effects 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 238000001514 detection method Methods 0.000 description 6
- 238000003209 gene knockout Methods 0.000 description 6
- 108091092878 Microsatellite Proteins 0.000 description 5
- 230000004913 activation Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 101150037123 APOE gene Proteins 0.000 description 4
- 238000011121 vaginal smear Methods 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 210000004508 polar body Anatomy 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000004291 uterus Anatomy 0.000 description 3
- 206010002091 Anaesthesia Diseases 0.000 description 2
- 102000013918 Apolipoproteins E Human genes 0.000 description 2
- 108010025628 Apolipoproteins E Proteins 0.000 description 2
- 102000009151 Luteinizing Hormone Human genes 0.000 description 2
- 108010073521 Luteinizing Hormone Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 210000001771 cumulus cell Anatomy 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 230000035935 pregnancy Effects 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 210000001215 vagina Anatomy 0.000 description 2
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 1
- NALREUIWICQLPS-UHFFFAOYSA-N 7-imino-n,n-dimethylphenothiazin-3-amine;hydrochloride Chemical compound [Cl-].C1=C(N)C=C2SC3=CC(=[N+](C)C)C=CC3=NC2=C1 NALREUIWICQLPS-UHFFFAOYSA-N 0.000 description 1
- 241001260012 Bursa Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
- 102000001974 Hyaluronidases Human genes 0.000 description 1
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 1
- BVIAOQMSVZHOJM-UHFFFAOYSA-N N(6),N(6)-dimethyladenine Chemical compound CN(C)C1=NC=NC2=C1N=CN2 BVIAOQMSVZHOJM-UHFFFAOYSA-N 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- JVHIPYJQMFNCEK-UHFFFAOYSA-N cytochalasin Natural products N1C(=O)C2(C(C=CC(C)CC(C)CC=C3)OC(C)=O)C3C(O)C(=C)C(C)C2C1CC1=CC=CC=C1 JVHIPYJQMFNCEK-UHFFFAOYSA-N 0.000 description 1
- ZMAODHOXRBLOQO-UHFFFAOYSA-N cytochalasin-A Natural products N1C(=O)C23OC(=O)C=CC(=O)CCCC(C)CC=CC3C(O)C(=C)C(C)C2C1CC1=CC=CC=C1 ZMAODHOXRBLOQO-UHFFFAOYSA-N 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000005059 dormancy Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229960002773 hyaluronidase Drugs 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- PGHMRUGBZOYCAA-ADZNBVRBSA-N ionomycin Chemical compound O1[C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)/C=C/C[C@@H](C)C[C@@H](C)C(/O)=C/C(=O)[C@@H](C)C[C@@H](C)C[C@@H](CCC(O)=O)C)CC[C@@]1(C)[C@@H]1O[C@](C)([C@@H](C)O)CC1 PGHMRUGBZOYCAA-ADZNBVRBSA-N 0.000 description 1
- PGHMRUGBZOYCAA-UHFFFAOYSA-N ionomycin Natural products O1C(CC(O)C(C)C(O)C(C)C=CCC(C)CC(C)C(O)=CC(=O)C(C)CC(C)CC(CCC(O)=O)C)CCC1(C)C1OC(C)(C(C)O)CC1 PGHMRUGBZOYCAA-UHFFFAOYSA-N 0.000 description 1
- 229960002725 isoflurane Drugs 0.000 description 1
- 210000000982 limb bud Anatomy 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 230000007420 reactivation Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0273—Cloned vertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/02—Animal zootechnically ameliorated
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Developmental Biology & Embryology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Animal Husbandry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to the method for somatic cell clone dog, more particularly, to the method for preparing somatic cell clone dog together with autologous embryo transfer technology using Hyposmolality fusion liquid.
Description
Technical field
The present invention relates to the method for improved somatic cell clone dog, more particularly, to using Hyposmolality fusion liquid together with
The method that autologous embryo transfer technology prepares somatic cell clone dog.
Background technology
Animal cloning technology is to be transferred to cell in receptor oocytes, production has with donorcells by nuclear transfer
There is the animal individual of identical DNA sequence dna information.The maximum feature of the technology is the cloned animal being born with thin with donor
The completely the same hereditary information of born of the same parents.Therefore animal somatic cell can be replicated using clone technology, clone technology can be applied to turn base
Because animal productiong, excellent domestic animal expand numerous, animals on the brink of extinction resource conservation, progress therapeutic cloning etc..
Dog is the important domestic animal of the mankind, acts not only as pet and is raised, while is also medicine and biology
Important large-scale model animal in research.Clone dog is produced using clone technology, pet dog can be not only cloned, be given birth to for the mankind
It is living to bring enjoyment, while using technology production transgenosis and Gene Knock-Out Animal Model model, will greatly promote medicine and biology
Development.
The first somatic cell clone dog succeeded in 2005, and the Research Team taught by Korean science man Huang Yuxi is complete
Into carrying out nuclear transfer, successful clone afghan hound " Snuppy " using the dog egg mother cell of cylinder mature.
Dog somatic cell clone technique can be applied not only to scientific research, and what is also obtained in terms of working dog clone widely should
With.The end of the year 2007,7 somatic cell clone police dogs of South Korea are born, this 7 Labrador retrievers are the first batch of somatic cell clones in the world
Police dog, be using a first place for " Chase " Labrador retriever somatic cell clone it is successful.These clone police dogs exist
Start to receive professional training soon after birth, and good characteristic essential to showing many drug detector dogs in training.2009
Year, Korean science every household utilize somatic cell clone technique, successful clone 5 U.S. " 911 event " hero police dog " Te La
Gram ", this 5 clone dogs inherit " Te Lake " excellent gene, have ability to work outstanding as " Te Lake ".
However, in the technology of existing somatic cell clone dog, the bitch of estrus synchronization is usually selected to carry out allosome embryo's shifting
Plant, success rate is relatively low, the increase of clone's dog production cost is caused, moreover, the required heat bitch quantity of allosome embryo transfer
More, operational means is complicated, it is difficult to realizes the application of industrialization.Hence it is highly desirable to improve existing somatic cell clone dog skill
Art, the somatic cell clone dog of high success rate is obtained with shirtsleeve operation means, less experimental dog and low cost, so that real
The commercial application of existing somatic cell clone dog.
The content of the invention
Present invention employs the fusion liquid of Hyposmolality, the fusion efficiencies of embryo are improved；And embryo transplantation acceptor dog
Only punching takes the egg mother cell in unilateral fallopian tubal, and egg mother cell obtains after nuclear transfer, fusion and activation with other donor dogs
The clone embryos obtained, co-transplantation enter not rush in another oviductus lateralis of ovum, realize the embryo transfer that autologous/allosome is combined,
Improve the pregnancy efficiency of clone dog.
In a first aspect, the present invention provides the method for somatic cell clone dog, described method includes following steps：(1) body is thin
Born of the same parents' is separately cultured；(2) enucleation oocyte is prepared from acceptor bitch；(3) body cell is imported to the cell of enucleation oocyte
In matter；(4) clone embryos are activated；(5) by the clone embryo transplantation that step (4) obtains to acceptor bitch；It is characterized in that：Go
Core egg mother cell is obtained from the fallopian tubal that the only unilateral acceptor bitch for rushing ovum rushes ovum；And in above-mentioned steps (5), embryo will be cloned
Tire is transplanted to acceptor bitch and is not rushed in that oviductus lateralis of ovum.
, it is necessary to which first identification has into the bitch in oestrus in step (2) prepares enucleation oocyte from acceptor bitch
For body, the vaginal smear of blood drawing detection daily, progesterone, promote ooecium element and lutropin, when keratinocyte ratio reaches 80-
More than 90%, when progesterone level reaches 4-7ng/mL or so, assert that bitch is in the onset of ovulation.The 72h-120h after ovulation, carries out
One side rushes ovum and obtains ripe egg mother cell, then carries out the stoning processing of mature oocyte.
Preferably, body cell is imported in the cytoplasm of enucleation oocyte using electro' asion in above-mentioned steps (3),
And the fusion liquid that osmotic pressure is 200mOSM-280mOSM is used in electro' asion.
Preferably, body cell is imported in the cytoplasm of enucleation oocyte using electro' asion in above-mentioned steps (3),
And the fusion liquid that osmotic pressure is 240mOSM is used in electro' asion.
The composition of the fusion liquid is as follows：0.2-0.28M mannitol, 0.1mM MgSO4, 0.5mM Hepes and 0.05%
BSA。
Preferably, the composition of the fusion liquid is as follows：0.24M mannitol, 0.1mM MgSO4, 0.5mM Hepes and
0.05%BSA.
Preferably, electro' asion uses the voltage of 2-4kv/cm.
The method of intracytoplasmic injection can also be used to import dog body cell in the kytoplasm of enucleation oocyte, in kytoplasm
Injection generally uses Piezo microinjection systems, directly in whole donorcells or donorcells core injection kytoplasm, is noted in kytoplasm
The method penetrated need not carry out electro' asion.
Second aspect, the present invention provides the method for somatic cell clone dog, described method includes following steps：(1) body is thin
Born of the same parents' is separately cultured；(2) enucleation oocyte is prepared from acceptor bitch and donor bitch；(3) body cell is imported into stoning ovum mother
In the cytoplasm of cell；(4) clone embryos are activated；(5) by the clone embryo transplantation that step (4) obtains to acceptor bitch；It is special
Sign is：A part of enucleation oocyte is obtained from the fallopian tubal that the only unilateral acceptor bitch for rushing ovum rushes ovum, another part stoning ovum
Mother cell is obtained from the bilateral salpingo that bilateral salpingo has carried out the donor bitch for rushing ovum；And in above-mentioned steps (5),
In the fallopian tubal that clone embryo transplantation is not rushed ovum to acceptor bitch.
, it is necessary to which first identification has into the bitch in oestrus in step (2) prepares enucleation oocyte from acceptor bitch
For body, the vaginal smear of blood drawing detection daily, progesterone, promote ooecium element and lutropin, when keratinocyte ratio reaches 80-
More than 90%, when progesterone level reaches 4-7ng/mL or so, assert that bitch is in the onset of ovulation.The 72h-120h after ovulation, carries out
Rush ovum and obtain ripe egg mother cell, then carry out the stoning processing of mature oocyte.
Preferably, body cell is imported in the cytoplasm of enucleation oocyte using electro' asion in above-mentioned steps (3),
And the fusion liquid that osmotic pressure is 200mOSM-280mOSM is used in electro' asion.
Preferably, body cell is imported in the cytoplasm of enucleation oocyte using electro' asion in above-mentioned steps (3),
And the fusion liquid that osmotic pressure is 240mOSM is used in electro' asion.
The composition of the fusion liquid is as follows：0.2-0.28M mannitol, 0.1mM MgSO4, 0.5mM Hepes and 0.05%
BSA。
Preferably, the composition of the fusion liquid is as follows：0.24M mannitol, 0.1mM MgSO4, 0.5mM Hepes and
0.05%BSA.
Preferably, electro' asion uses the voltage of 2-4kv/cm.
The method of intracytoplasmic injection can also be used to import dog body cell in the kytoplasm of enucleation oocyte, in kytoplasm
Injection generally uses Piezo microinjection systems, directly in whole donorcells or donorcells core injection kytoplasm, is noted in kytoplasm
The method penetrated need not carry out electro' asion.
The body cell of the present invention can come from various tissues and organ, such as fetal tissue, skin, muscle, ear, breast
The cell of gland, fallopian tubal, ovary, blood, urine, fat, marrow, blood vessel, tube chamber endothelium etc..Available for the method for the present invention
The example of body cell includes but is not limited to：Fetal fibroblast, Skin Cell, epithelial cell, ear cell, into fiber finer
Born of the same parents, endothelial cell, muscle cell, mammary glandular cell, oviduct cell, gonad cell, cumulus cell, nerve cell, osteoblast
Deng.
From the embodiment above as can be seen that acceptor bitch also provides egg mother cell in itself in the method for the present invention, and
It is only unilateral fallopian tubal to be carried out rushing ovum to obtain egg mother cell；And the clone that will be obtained through nuclear transfer, electro' asion and activation
In that oviductus lateralis that embryo transfer does not rush ovum to acceptor bitch, therefore, above two technical solution necessarily refers to autologous shifting
Plant, the usage amount of experimental dog is greatly reduced compared with heteroplastic transplantation in the prior art needs heat bitch quantity more.It is existing
Have in technology, it usually needs the dog of more than 40.The method of the present invention only needs several experimental dogs.
Moreover, need to select the bitch of estrus synchronization to carry out allosome embryo transfer in the prior art, but bitch heat is reflected
It is fixed more difficult, synchronization is carried out by heat and identifies that accuracy rate is relatively low, this is also the original for causing allosome Success Rate of Embryo Transfer relatively low
Cause.Comparatively speaking, the autotransplantation necessarily included in technical scheme is largely avoided to be carried out by heat
The step of synchronization judges；Meanwhile autotransplantation can increase substantially the implantation efficiency of clone embryos, it is achieved thereby that drop
The target of dog is cloned in the production cost of low clone dog and efficiently production.
In addition, the present invention uses fusion liquid of the osmotic pressure for 200-280mOSM, it is 280- with osmotic pressure in the prior art
The fusion liquid phase ratio of 310mOSM, significantly improves the fusion efficiencies of embryo.
Abbreviation and Key Term definition：
Clone：Using corresponding technological means, the animal individual that there is identical DNA sequence dna with donor cell is produced.
NT：Dog adult cell, is implanted into the side that clone embryos are built in the dog egg mother cell of stoning by body-cell neucleus transplanting
Method.
DC：Donor cell, includes the cell of complete inhereditary material, is implanted into receptor oocytes and be used for
Prepare somatic cell clone animal.
AET：Autologous embryo transfer, after heat bitch goes out MII egg mother cells progress body-cell neucleus transplanting, then to rush ovum
Acceptor of the bitch as clone embryo transplantation, prepares somatic cell clone dog.
Brief description of the drawings
Fig. 1 is the clone dog that APOE gene knockouts dog-apple (Figure 1A) that numbering is 161207 is 170502 with numbering
The contrast photo that " dragon dragon " (Figure 1B) is born 30 days respectively..
Embodiment
Technical scheme is described further with reference to embodiment and Figure of description.These embodiments
It is merely to illustrate the present invention rather than limits protection scope of the present invention.
Embodiment：
(1) body cell is separately cultured
In this experiment, the donor dog of body cell for the raising of Beijing Xi Nuo paddy bio tech ltd experiment with than lattice
Dog-apple (numbering 161207), is world's the first APOE gene knockouts prepared by Beijing Xi Nuo paddy bio tech ltd
(Classification And Nomenclature with its whole hereditary information is apo E (APOE) gene knockout beasle dog ear fibroblast to dog：
BGD-APOEKO-EF0 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation
Location is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 (postcode 100101), preserving number are CGMCC No.13804, are protected
The Tibetan date is on March 1st, 2017).When taking skin histology, wiped off first with grainer around the dog ear-edge tissue to be gathered
Fur, using 75% alcohol disinfecting, with the ear-edge tissue about 1cm of the scissors clip dog after sterilizing2, then insert containing
In the DMEM basal mediums of the penicillin+100IU/ml streptomysins of 100IU/ml, in taking back laboratory in 12h.
Tissue block is recycled containing 5 times dual anti-(penicillin and streptomysin) first with PBS cleaning 3 times or more than 3 times
DMEM culture mediums clean 3 times, carefully remove adipose tissue with eye scissors, achieve the purpose that to expose skin corium.Skin histology is turned
Into another sterile petri dish, sheared using scalpel and be cut into l mm2The tissue block of size.With ophthalmic tweezers by tissue
Block moves to the ware bottom of culture dish, is spread and is dissipated uniformly with pipette tips, then overturn blake bottle, add containing 20% FBS and 5
Dual anti-DMEM nutrient solutions again, it is 37 DEG C, 5%CO to be put into environment2, 100% humidity incubator in cultivate.After when 7-8 is small,
Carry out turning over bottle after tissue block is docile and obedient, make culture medium continue to cultivate after being totally submerged tissue block, it is every 48 it is small when replace nutrient solution one
It is secondary.
(2) bitch detection of oestrus
Oocyte donors and acceptor dog 16 are used in the present embodiment altogether, detection of oestrus uses vaginal smear and serum
Progesterone detects.Into the bitch in oestrus, vaginal smear detection is carried out daily, uses swab stick physiological saline of the length for 5cm
Vagina is inserted into after infiltration, extract is then applied to glass slide, after being dyed with Giemsa stain, counts vagina under the microscope
Epithelial cell angling degree, angling degree think that it is in the onset of ovulation when being more than 80%.When progesterone detects, 3mL blood is extracted,
Serum is centrifuged, progesterone content is detected using progesterone detecting instrument, is to think its row of being in when progesterone value reaches 4-7ng/mL
The ovum phase, 72-120h is rushed and is taken mature oocyte after ovulation.
(3) acquisition of mature oocyte
Bitch anaesthetizes the precious induced anesthesia of dog dormancy first by 0.1mL, then maintains fiber crops using isoflurane and lung ventilator
It is liquor-saturated.Exposure ovary and the palace pipe engaging portion in uterus, the metal injection pin with circular front end is inserted at the breach of ovarian bursa
Fimbriae tubae portion, then fixes needle tubing；Injection needle is inserted at the fallopian tubal of palace pipe engaging portion, with containing 10%FBS's
TCM199 culture mediums rinse fallopian tubal, and the plastic tube that length is 3cm is punctured into inside uterus, it is fixed after using egg liquid from
Egg mother cell is rinsed in condominium engaging portion.Rushing ovum direction also can inversely carry out, but the needle tubing diameter for picking up egg liquid one end is greater than
The needle tubing diameter that egg liquid enters.The bitch for electing acceptor as only carries out the ovum that rushes of an oviductus lateralis, and opposite side is left embryo
Transplanting is prepared.After rushing ovum, bitch carries out revival processing immediately, and when embryo transfer is anaesthetized again.Other only provide ovum
The donor dog of mother cell then carries out the ovum that rushes of bilateral salpingo, and the egg mother cell of acquisition is used for body-cell neucleus transplanting.
(4) oocyte enucleation
The inner or in vitro ripe egg mother cell of collection is put into the DPBS solution containing 0.1% hyaluronidase,
In 37 DEG C of thermal station, blown and beaten repeatedly using liquid-transfering gun, cumulus cell is removed.Under inverted microscope, amplify 200 times of observations,
To select the mature oocyte containing first polar body.
By the egg mother cell of select, be put into HEPES bufferings containing 10%FBS and 5ug/ml cytochalasin Bs
In CR2aa micromanipulation liquid, 30min is incubated, softens its cytoskeleton, then using injection needle by first polar body, phase
Adjacent cytoplasm (being less than 5%) and oocyte nuclei remove, and then the enucleation oocyte is stored in SOF culture mediums.Will
The first polar body and kytoplasm that injection needle suctions out, are put into Hochest33342 solution and dye, then under fluorescence microscope
Whether the suctioned out kytoplasm of observation contains nucleus, judges Enucleation efficiency.If more than 90%, it is thin can to carry out body for stoning rate
Born of the same parents inject, and otherwise egg mother cell will be dyed using Hochest33342, ultraviolet irradiation is carried out under fluorescence microscope,
Indicated nucleus is removed clean.
(5) enucleation oocyte note core, fusion and activation
Enucleation oocyte is placed in the CR2aa micromanipulation liquid of no CB, is then expelled to the donor cell of selection
Between egg mother cell oolemma and cytoplasm, oolemma is gently pressed with injection needle, body cell is closely tied with oocyte membrane
Close structure reconstructed embryo.Reconstructed embryo is placed in the fusion liquid that osmotic pressure is 240mOSM and (includes 0.24M mannitol, 0.1mM
MgSO4, 0.5mM Hepes and 0.05%BSA) in, it is then placed between the parallel pole of integration slot, using ECM2001
(BTX) electro' asion is carried out under the conditions of 2-4kv/cm voltages, 2 subpulses, 10 μ s of pulse spacing.30min is in body formula after fusion
The fusion situation of micro- Microscopic observation oocyte cytoplasm and donor cell, counts fusion efficiencies.It is determined as the reconstruct of fusion
The ionomycin activation 4min of 10 μm of ol/L of embryo, then uses the mSOF reactivation 4h of the 6-DMAP containing 2mmol/L, completes
Embryo transfer is ready for after activation.Referring to following table one, it can be seen that during embryo fusion, using melting for 240mOsm osmotic pressure
Liquid is closed, fusion rate can be maintained more than 70%；And the fusion liquid for commonly using osmotic pressure (300mOsm) in the prior art is used, melt
Conjunction rate is only 50% or so.
The influence of table one, fusion liquid osmotic pressure to fusion rate
| Merge liquid osmotic pressure (mOsm) | Shock by electricity embryo number | Merge embryo number | Fusion rate |
| 240 | 61 | 45 | 73% |
| 300 | 43 | 7 | 53.4% |
(6) embryo transfer
After the anesthesia of acceptor dog, the ovary tissue that ovum is not rushed in the stomach wall exposure of ovum side is not rushed in selection, and uterus and ovary are led
Go out.Clone embryos are sucked in embryo transplantation tube, embryo transplantation tube are inserted into from fimbriae tubae portion, by Embryonic limb bud cell acceptor body.
Referring to table two, B ultrasound detection is carried out within 25 days after embryo transfer, embryo transfer is the results show that using certainly/allosome embryo transfer mode
Pregnancy rate, this experiment co-transplantation acceptor 4, wherein 2 total 3 clones dogs that are pregnant and give birth to is greatly improved.
Table two, embryo transfer collect
(7) microsatellite and the identification of APOE gene knockouts of dog are cloned
To determine birth clone puppy and somatic cell donor dog-" apple " (numbering：161207) whether there is identical something lost
Communication ceases, and determines the affiliation with embryo transplantation acceptor bitch, and collection clone puppy and replace-conceive bitch blood, use
The method of microsatellite identification identifies triangular genetic affinity, selects 14 microsatellite locus, is respectively：PEZ2、PEZ3、
PEZ5, PEZ6, PEZ8, PEZ12, PEZ15, PEZ20, PEZ21, FH2011, FH2054, FH2079, FH2132, FH2611 and
VWFX, this identification are carried out by Ministry of Public Security Nanchang police dog base DNA laboratories.
In first identification, the censorship sample for the apple for being 161207 by the use of numbering as No. 1 sample, using by by
The clone puppy dragon dragon that the numbering that body dog NTR1217 is born is 170502 is used as No. 2 samples, using acceptor dog NTR1217 conducts
No. 3 samples, identify the homogeneity of No. 2 samples and No. 1 sample, and identify the parent child relationship of No. 2 samples and No. 3 samples.
Blood sample is equally divided into two parts, does parallel control, and PCR amplification is carried out using dog STR fluorescence detection reagent kits.
Amplified production carries out electrophoresis and parting using AB131030 genetic analyzers.
STR polymorphism inspection results：By comparing, No. 2 dog samples are consistent with No. 1 dog sample STR parting；No. 2 dogs and 3
Number dog sample is mismatched in multiple locus such as PEZ2, FH2054, FH2132 and PEZ15.
In second identification, the censorship sample for the apple for being 161207 by the use of numbering as No. 2 samples, using by by
The clone puppy that the numbering that body dog NTR1243 is born is 170610 is as No. 3 samples, the numbering born by acceptor dog NTR1243
For 170611 clone puppy as No. 4 samples, using acceptor dog NTR1243 as No. 1 sample, No. 3, No. 4 samples of identification with
The homogeneity of No. 2 samples, and identify the parent child relationship of No. 3 samples and No. 1 sample.
Two parts are taken from every part of sample with sampler, does parallel control, is carried out using dog STR fluorescence detection reagent kits
PCR amplification.Amplified production carries out electrophoresis and parting using AB131030 genetic analyzers.
STR polymorphism inspection results：By comparing, No. 3, No. 4 samples and No. 2 sample locus Data Matchings；No. 3,4
Number sample and No. 1 sample are mismatched in multiple locus such as PEZ12, PEZ15.
Qualification result shows, the clone dog and numbering that clone dog " dragon dragon " that numbering is 170502, numbering are 170610
The cell donor dog " apple " that clone dog for 170611 is 161207 with numbering, is in all microsatellite locus partings
Unanimously, and with embryo transplantation acceptor dog NTR1217 and NTR1243, PEZ2, PEZ5, PEZ6, PEZ8, PEZ12, PEZ15,
The sites such as PEZ20, FH2011, FH2054, FH2079, FH2132, FH2611 and VWFX are mismatched (referring to following table three), it was demonstrated that
The clone dog that the clone dog and numbering that clone dog that numbering is 170502 " dragon dragon ", numbering are 170610 are 170611 is volume
Number for 161207 cell donor dog " apple " clone dog.Attached drawing 1 is APOE gene knockouts dog-apple that numbering is 161207
The contrast photo that the clone dog " dragon dragon " (Figure 1B) that fruit (Figure 1A) is 170502 with numbering is born 30 days respectively.
Table three, clone's dog microsatellite qualification result
Claims (18)
1. the method for somatic cell clone dog, described method includes following steps：(1) body cell is separately cultured；(2) it is female from acceptor
Dog prepares enucleation oocyte；(3) body cell is imported in the cytoplasm of enucleation oocyte；(4) clone embryos are activated；(5)
By the clone embryo transplantation that step (4) obtains to acceptor bitch；It is characterized in that：Enucleation oocyte is obtained from only one side and rushes ovum
Acceptor bitch rushes the fallopian tubal of ovum；And in above-mentioned steps (5), clone embryo transplantation that side of ovum is not rushed into acceptor bitch
In fallopian tubal.
2. the method according to claim 1, in step (2) from acceptor bitch prepares enucleation oocyte, identifies keratinocyte
Ratio reaches more than 80-90%, progesterone level reach the bitch of 4-7ng/mL or so as the bitch in the onset of ovulation for use as
Acceptor bitch.
3. method according to claim 2, the 72h-120h after ovulation, carry out one side and rush the egg mother cell that ovum obtains maturation, so
The stoning processing of mature oocyte is carried out afterwards.
4. according to the method for any one of claim 1-3, it is characterised in that using electro' asion that body is thin in above-mentioned steps (3)
Born of the same parents are imported in the cytoplasm of enucleation oocyte, and the fusion liquid that osmotic pressure is 200mOSM-280mOSM is used in electro' asion.
5. according to the method for any one of claim 1-3, it is characterised in that using electro' asion that body is thin in above-mentioned steps (3)
Born of the same parents are imported in the cytoplasm of enucleation oocyte, and the fusion liquid that osmotic pressure is 240mOSM is used in electro' asion.
6. method according to claim 4, the composition of the fusion liquid is as follows：0.2M-0.28M mannitol, 0.1mM MgSO4,
0.5mM Hepes and 0.05%BSA.
7. according to the method for any one of claim 4-6, it is characterised in that electro' asion uses the voltage of 2kv/cm-4kv/cm.
8. according to the method for any one of claim 1-7, the body cell is from following tissue or organ：Fetal tissue, skin
Skin, muscle, ear, mammary gland, fallopian tubal, ovary, blood, urine, fat, marrow, blood vessel and tube chamber endothelium.
9. according to the method for any one of claim 1-7, the body cell is selected from：Fetal fibroblast, Skin Cell, epithelium
Cell, ear cell, fibroblast, endothelial cell, muscle cell, mammary glandular cell, oviduct cell, gonad cell, ovarian cumulus are thin
Born of the same parents, nerve cell and osteoblast.
10. the method for somatic cell clone dog, described method includes following steps：(1) body cell is separately cultured；(2) from acceptor
Bitch and donor bitch prepare enucleation oocyte；(3) body cell is imported in the cytoplasm of enucleation oocyte；(4) activate
Clone embryos；(5) by the clone embryo transplantation that step (4) obtains to acceptor bitch；It is characterized in that：Part stoning ovum is female thin
Born of the same parents are obtained from the fallopian tubal that the only unilateral acceptor bitch for rushing ovum rushes ovum, another part enucleation oocyte be obtained from bilateral salpingo into
The bilateral salpingo of the donor bitch for rushing ovum is gone；And in above-mentioned steps (5), by clone embryo transplantation to acceptor bitch not
Rush in the fallopian tubal of ovum.
11. method according to claim 10, in step (2) from acceptor bitch prepares enucleation oocyte, identification angling is thin
Born of the same parents' ratio reaches more than 80-90%, progesterone level reach the bitch of 4-7ng/mL or so as the bitch in the onset of ovulation with
Make acceptor bitch.
12. method according to claim 11, the 72h-120h after ovulation, carry out one side and rush the egg mother cell that ovum obtains maturation,
Then the stoning processing of mature oocyte is carried out.
13. according to the method for any one of claim 10-12, it is characterised in that electro' asion is used in above-mentioned steps (3) by body
Cell is imported in the cytoplasm of enucleation oocyte, and the fusion that osmotic pressure is 200mOSM-280mOSM is used in electro' asion
Liquid.
14. according to the method for any one of claim 10-12, it is characterised in that electro' asion is used in above-mentioned steps (3) by body
Cell is imported in the cytoplasm of enucleation oocyte, and the fusion liquid that osmotic pressure is 240mOSM is used in electro' asion.
15. method according to claim 13, the composition of the fusion liquid is as follows：0.2M-0.28M mannitol, 0.1mM MgSO4,
0.5mM Hepes and 0.05%BSA.
16. according to the method for any one of claim 13-15, it is characterised in that electro' asion uses the voltage of 2kv/cm-4kv/cm.
17. according to the method for any one of claim 10-16, the body cell is from following tissue or organ：Fetal tissue,
Skin, muscle, ear, mammary gland, fallopian tubal, ovary, blood, urine, fat, marrow, blood vessel and tube chamber endothelium.
18. according to the method for any one of claim 10-16, the body cell is selected from：Fetal fibroblast, Skin Cell,
Epithelial cell, ear cell, fibroblast, endothelial cell, muscle cell, mammary glandular cell, oviduct cell, gonad cell, ovum
Mound cell, nerve cell and osteoblast.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710614323.3A CN107937444A (en) | 2017-07-25 | 2017-07-25 | The method of somatic cell clone dog |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710614323.3A CN107937444A (en) | 2017-07-25 | 2017-07-25 | The method of somatic cell clone dog |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107937444A true CN107937444A (en) | 2018-04-20 |
Family
ID=61928594
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710614323.3A Pending CN107937444A (en) | 2017-07-25 | 2017-07-25 | The method of somatic cell clone dog |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107937444A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108977408A (en) * | 2018-08-14 | 2018-12-11 | 北京希诺谷生物科技有限公司 | The method for preparing dog in vitro fertilization using maturation in vitro dog egg mother cell |
| CN109280637A (en) * | 2018-10-26 | 2019-01-29 | 安徽农业大学 | A kind of method of efficient removal pig immature oocyte surrounding cumulus cells |
| CN110656183A (en) * | 2018-06-28 | 2020-01-07 | 深圳市华大司法技术协同创新研究院 | STR locus set for dogs and application |
| CN116640804A (en) * | 2023-04-20 | 2023-08-25 | 云南农业大学 | Method for constructing triploid pig based on somatic cell nuclear transfer technology |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101225382A (en) * | 2007-01-17 | 2008-07-23 | 首尔大学校产学协力财团 | Method for producing cloned dog |
| CN101228265A (en) * | 2005-07-26 | 2008-07-23 | 首尔大学校产学协力财团 | Cloned canines and method for producing thereof |
| CN101668847A (en) * | 2007-11-19 | 2010-03-10 | 财团法人首尔大学校产学协力财团 | Methods for improvement of birth rates in canidae on somatic cell nuclear transfer |
-
2017
- 2017-07-25 CN CN201710614323.3A patent/CN107937444A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101228265A (en) * | 2005-07-26 | 2008-07-23 | 首尔大学校产学协力财团 | Cloned canines and method for producing thereof |
| CN101225382A (en) * | 2007-01-17 | 2008-07-23 | 首尔大学校产学协力财团 | Method for producing cloned dog |
| CN101668847A (en) * | 2007-11-19 | 2010-03-10 | 财团法人首尔大学校产学协力财团 | Methods for improvement of birth rates in canidae on somatic cell nuclear transfer |
Non-Patent Citations (3)
| Title |
|---|
| BYEONG CHUN LEE: "Dogs cloned from adult somatic cells", 《NATURE》 * |
| KIM, MJ: "Influence of oocyte donor and embryo recipient conditions on cloning efficiency in dogs", 《THERIOGENOLOGY》 * |
| Q ZOU: "Generation of gene-target dogs using CRISPR/Cas9 system", 《JOURNAL OF MOLECULAR CELL BIOLOGY》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110656183A (en) * | 2018-06-28 | 2020-01-07 | 深圳市华大司法技术协同创新研究院 | STR locus set for dogs and application |
| CN108977408A (en) * | 2018-08-14 | 2018-12-11 | 北京希诺谷生物科技有限公司 | The method for preparing dog in vitro fertilization using maturation in vitro dog egg mother cell |
| CN109280637A (en) * | 2018-10-26 | 2019-01-29 | 安徽农业大学 | A kind of method of efficient removal pig immature oocyte surrounding cumulus cells |
| CN116640804A (en) * | 2023-04-20 | 2023-08-25 | 云南农业大学 | Method for constructing triploid pig based on somatic cell nuclear transfer technology |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107937444A (en) | The method of somatic cell clone dog | |
| Lonergan | Historical and futuristic developments in bovine semen technology | |
| CN103205463B (en) | Nuclear transplantation | |
| CN106520838A (en) | New method for gene injection for somatic cell nuclear transfer reconstructed embryo | |
| CN105524940A (en) | Vector, cell and method for improving bovine cloning efficiency on the basis of histone methylation modifying level | |
| Morohaku et al. | Development of fertile mouse oocytes from mitotic germ cells in vitro | |
| US20190032086A1 (en) | Method for preparing a gene knock-out canine with somatic cell cloning technology | |
| Cao et al. | Effects of chemically defined medium on early development of porcine embryos derived from parthenogenetic activation and cloning | |
| CN108060117B (en) | Method for improving pig cloned embryo development efficiency | |
| CN104513807B (en) | The method for cloning non-human animal is separated, cultivates the method for cell and carried out from blood | |
| CN102517329A (en) | Method for breeding transgenic pig for over expression of pig PBD-2 gene | |
| Moustafa et al. | The fate of transplanted cells in mouse blastocysts in vitro | |
| Yu et al. | Early developmental processes in the fertilised honeybee (Apis mellifera) oocyte | |
| CN101580828B (en) | Nuclear transfer method | |
| Liu et al. | Fibroblasts from the new-born male testicle of Guangxi Bama mini-pig (Sus scrofa) can support nuclear transferred embryo development in vitro | |
| KR20080077738A (en) | A methods to improve in vitro maturation of pig oocytes | |
| KR101046706B1 (en) | Straw Embryos Implantation in Mammals | |
| Lee et al. | Nuclear transfer in ruminants | |
| CN105002157A (en) | Porcine somatic cell nucleus transplanting fusion method | |
| CN109234225A (en) | A kind of embryo medium and a kind of processing method of somatic cell clone embryo | |
| CN110904034A (en) | Method for removing egg cell nucleus | |
| CN103509752B (en) | Hubei white pig fetal fibroblast cell line | |
| CN103710386B (en) | The preparation method of transgenic animal | |
| CN103409467B (en) | Method for acquiring heterogeneity interspecies-cloned yak premium embryos | |
| CN103952424B (en) | Method for producing double-muscular trait somatic cell cloned pig with MSTN (myostatin) bilateral gene knockout |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |