CN107227298A - A kind of clone embryos treatment fluid and its application method and the purposes of the treatment fluid - Google Patents
A kind of clone embryos treatment fluid and its application method and the purposes of the treatment fluid Download PDFInfo
- Publication number
- CN107227298A CN107227298A CN201710339889.XA CN201710339889A CN107227298A CN 107227298 A CN107227298 A CN 107227298A CN 201710339889 A CN201710339889 A CN 201710339889A CN 107227298 A CN107227298 A CN 107227298A
- Authority
- CN
- China
- Prior art keywords
- cell
- embryo
- treatment fluid
- clone embryos
- 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.)
- Granted
Links
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
- 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
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
-
- 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
- C12N5/0608—Germ cells
- C12N5/0609—Oocytes, oogonia
-
- 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
- C12N2500/05—Inorganic components
-
- 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
-
- 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
- C12N2500/32—Amino acids
-
- 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
- C12N2500/32—Amino acids
- C12N2500/33—Amino acids other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Reproductive Health (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Gynecology & Obstetrics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of clone embryos treatment fluid, it is made up of BIX 01294 and PZM nutrient solutions, BIX 01294 concentration is 5nM~500nM in PZM nutrient solutions；Wherein, PZM nutrient solutions are composed of the following components：NaCl 3.156g；NaHCO31.053g；KCl 0.373g；KH2SO40.024g；MgSO40.049g；Calcium lactate 0.308g；Pyruvic acid 0.011g；L glutamic acid 0.073g；Hypotaurine 0.273g；BME 10mL；MEM 5mL；Penicillin 0.033g；Streptomysin 0.025g；Sigma water, which is supplied, is settled to 500mL.Clone embryos are carried out with culture processing using the clone embryos treatment fluid of above technical scheme can reach following beneficial effect：(1) cell SUV39H1, SETdb1, DNMT1 or DNMT3a gene expression can be significantly reduced；(2) 2cell, the 4cell in early embryo development stage H3K9me2 levels can substantially be lowered.
Description
Technical field
The present invention relates to somatic cell clone technique, more particularly to a kind of clone embryos treatment fluid and its application method and should
The purposes for the treatment of fluid.
Background technology
Somatic cell clone (somatic cell nuclear transfer, SCNT), also known as somatic cell clone, are to be used for
Produce a kind of auxiliary procreation technology of animal.SCNT refers to donor somatic being transplanted to the cytoplasm of non-nucleus egg mother cell
In, by merging and activating so as to obtain reconstructed embryo, cell is recovered division, continue development and obtain complete with donorcells genotype
Exactly the same offspring.
The nucleus of the body cell of single differentiation can be cultivated in ex vivo environment and obtained by somatic cell clone technique
Whole living organism, this has huge potentiality (Yang for agricultural technology, biological medicine industry, endangered species protection
et al.,2007).In animal husbandry, nuclear donor progress animal cloning can be used as by setting up the cell line of excellent domestic animal, kept away
Exempt from the limitation for the animal breeding cycle and fertility efficiency for choosing seeds suffered under natural conditions, greatly shorten the breeding time limit, animal
The process of breeding accelerates therewith, promotes excellent drove to breed.In biomedicine, for cloned livestock and the body of transgenic animals
Cell clone technology is very valuable (Meurens et al., 2012).Cloned stem cell constitutes phase after induction differentiation
The histoorgan answered, transgenic animals work is produced for tissue or the donor of organ transplant, or by somatic cell clone technique
To produce the bioreactor of human medicine albumen.It can be used for preparing genetically modified animal by somatic cell clone technique, body is thin
Born of the same parents' clone technology has turned into the production that current producer gene modifies the technical way, the particularly big animal of transgenosis of animal,
Such as turn of the transgenic goat (Feng et al., 2015) of production expression human alpha-lactalbumin and expressing green fluorescent protein
Gene pig (Hyun et al., 2003) and ox (Arat et al., 2001；Rosen et al.,1999).By body cell gram
Grand technology saves endangered rareness species, can preserve body cell by the way that freezing is long-term, and recover when needing, this pole
It is big to improve the simplicity of protection rareness species, and cost is reduced, play important work in terms of species diversity is protected
With, and be expected to increase the amount of survival of rareness species using the technology.
Now, dedifferente reprogramming focuses primarily upon how to utilize body cell for the research of pluripotent state for body cell
Producing embryonic stem cell-like is used for regenerative therapies.Embryonic stem cell (embryonic stem cell, ESc) has complete
Totipotency and all cell types (Buehr et al., 2008 can be divided into；Evans et al., 1981), or even
Placenta cells (Murakami et al., 2011) are divided under Incubation Condition.The chromatin Structure of their high vigor
(Gaspar-Maia et al., 2011) and active chromosome transcriptional activity (Efroni et al., 2008) make them different
In the body cell (Meissner, 2010) of differentiation.But, from the perspective of from broader aspect, ESc seems to be considered as a kind of body thin
Born of the same parents, because they have some key characters with the embryo after other body cells or transplanting.Because the ESc of versatility can conduct
Vitro disease is modeled and the valuable cell derived of cell/tissue alternative medicine (Yang et al., 2007), and it can be from
(Wakayama et al., 2001) is set up in somatic cell clone technique, therefore, the somatic cell clone for human treatment learns
Technology is a kind of up-and-coming technology (Hochedlinger et al., 2003).DAHL etc. (Dahl et al., 2010) is
It is verified during ES is cell-derived, it causes istone lysine H3K4 tri-methylated (H3K4me3) and H3K27 front threes
Significant change occurs for base (H3K27me3), highlights epigenetic reprogramming relevant with the derivative of embryonic stem cell.Just because of
This advantage, carries out somatic cell clone using ESc, often there is higher cloning efficiency, but its efficiency is not still high.
The content of the invention
It is an object of the present invention to provide a kind of clone embryos treatment fluid, another purpose is to provide a kind for the treatment of fluid
Application method, also one purpose is to provide the purposes of the treatment fluid.
According to an aspect of the invention, there is provided a kind of clone embryos treatment fluid, by BIX-01294 and PZM nutrient solutions
Constitute, BIX-01294 concentration is 5nM~500nM in PZM nutrient solutions；Wherein, PZM nutrient solutions are composed of the following components：
In some embodiments, clone embryos treatment fluid pH is 7.2~7.4, and osmotic pressure is 288 ± 2mOsm.
In some embodiments, BIX-01294 concentration is 50nM~500nM.
According to another aspect of the present invention there is provided a kind of application method of above-mentioned clone embryos treatment fluid, including with
Lower step：
(1) pig ovary is collected, is put into the vacuum flask containing 1% 28-39 DEG C of dual anti-physiological saline, first with 39 DEG C
Physiological saline is cleaned up, and the egg mother cell in a diameter of 4-6mm ovarian follicles is then extracted with the 10ml syringes of No. 18 syringe needles of band,
Extract stands 15min in 39 DEG C of water-baths, goes after supernatant to add DPBS resuspensions, goes supernatant to lay equal stress on after standing 2-3min
It is multiple once, finally pick out that cytoplasm is uniform, ovarian cumulus is fine and close under Stereo microscope and cumulus cell-ovum of more than 2 layers of parcel
Mother cell complex is washed after 3 times respectively with DPBS and M199 maturation culture solutions, is transferred to and 2h has been balanced in incubator and has been covered
Cover in the M199 maturation cultures drop of embryo's level mineral oil or in four orifice plates, in 39 DEG C, the culture of 5%CO2 saturated humidities
Maturation culture 42h in case.
(2) cumulus cell-oocyte complex of maturation culture is transferred to the centrifuge tube containing hyaluronidase
In, it is transferred to after pipettor piping and druming about 2min in 30mm culture dishes, the egg mother cell for sloughing ovarian cumulus is sorted out with mouth suction pipe, washed
Egg mother cell, the mature oocyte with first polar body is chosen under body formula mirror.
(3) by the mature oocyte, with 100-120 μm of fixed pin of external diameter, internal diameter is adopted for 15-20 μm of kernel removing needle
It is enucleated, is comprised the following steps that with blind suction method：Drop 4 drips the operation drop of 50 μ L sizes in 65mm sterile culture disks, uses paraffin
Oil covering, then the egg mother cell of 30 or so and appropriate body cell are moved into wherein, ovum is gently fixed with fixed pin female thin
Born of the same parents, egg mother cell is stirred with kernel removing needle makes polar body in the position at general 5 o'clock, is then lunged, gone along 3 positions with kernel removing needle
Except polar body and neighbouring kytoplasm, then pin is withdrawn from, polar body and kytoplasm are spued, a size is subsequently injected into properly, surface light
Sliding body cell, completes embryo's restructuring procedure.Reconstructed embryo is put into renewal cultivation 1h in embryo medium；
(4) reconstructed embryo is transferred in fusion liquid after balance 2min in batches, then 5~8 every batch are put into and have been paved with
In the integration slot for merging liquid, reconstructed eggs are stirred with solid glass pin, make the cell membrane contact of donor cell and receptor oocytes
Face and electrode runs parallel, apply 150v/mm, 100 μ s, 2DC electric pulse induced fusion, activation reconstruct embryo then use embryo
Tire nutrient solution is transferred in the embryo medium of mineral oil covering immediately after washing 3 times, be placed in 39 DEG C, 5%CO2, saturated humidity
Cultivated in incubator.
(5) wash with nutrient solution the reconstructed embryo 5 times of fusion, be transferred in the good nutrient solution of pre-equilibration, be placed in 39 DEG C, saturation it is wet
Cultivated in degree, the incubator of hypoxia condition, continuation culture is carried out to embryo using the clone embryos treatment fluid.
In some embodiments, the hypoxia condition in above-mentioned steps (5) refers to that the air composition in the incubator is
5%O2+ 5%CO2+ 90%N2。
In some embodiments, it is further comprising the steps of before above-mentioned steps (5)：
After recovery cell, when cell confluency degree reaches more than 95%, the DMEM medium culture mistakes without serum are used instead
Night.Secondary daily digesting, operates liquid that the cell after centrifugation is resuspended into piping and druming uniformly, prepares clone with HN.
According to another aspect of the present invention there is provided above-mentioned clone embryos treatment fluid reduction cell SUV39H1,
Application in terms of SETdb1, DNMT1 or DNMT3a gene expression.
According to a further aspect of the invention the early embryo development stage is being lowered there is provided above-mentioned clone embryos treatment fluid
2cell, 4cell H3K9me2 levels application.
Clone embryos are carried out with culture processing using the clone embryos treatment fluid of above technical scheme can reach following have
Beneficial effect：
(1) cell SUV39H1, SETdb1, DNMT1 or DNMT3a gene expression can be significantly reduced；
(2) 2cell, the 4cell in early embryo development stage H3K9me2 levels can substantially be lowered.
Brief description of the drawings
Fig. 1 illustrates for influence of the clone embryos treatment fluid to cellular histone methylase related gene expression in the present invention
Figure.
Fig. 2 is influence schematic diagram of the clone embryos treatment fluid in the present invention to cell DNA methylase related gene expression.
Fig. 3 is clone embryos treatment fluid treatment group in the present invention and control group 2cell, 4cell immunofluorescence results figure.
Fig. 4 illustrates for clone embryos treatment fluid in the present invention to embryo's histone methylase related gene expression level
Figure.
Fig. 5 is clone embryos treatment fluid in the present invention to embryo's DNA methylation enzyme related gene expression level schematic diagram.
Embodiment
The invention will now be described in further detail with reference to the accompanying drawings.
Experiment material
1st, cell line and porcine clone embryos
Pig adult fibroblast (513D) picks up from the offer of Guangdong Wen Shi boars Science and Technology Ltd. water platform original seed pig farm
Boar；Porcine clone embryos are prepared in Guangdong Wen Shi boars Science and Technology Ltd. cloning experimentation room.
2nd, main agents, consumptive material
RNA extraction agent box E.Z.N.A.Total RNA Kit I are purchased from OMEGA companies；Embryo's RNA extraction agent boxes
Qiagen RNeasy Plus Micro Kit (50) are purchased from Qiagen companies；Reverse transcription reagent box PrimeScriptTMRT
Reagent Kit with gDNA Eraser are purchased from TaKaRa companies；Real-time quantitative PCR kit SYBR Select
Master Mix are purchased from Life Technologies companies；Regular-PCR is purchased from Guangzhou Dongsheng biotechnology company with Taq enzyme；
DL500DNA Marker are purchased from Dalian Bao Sheng bioengineering Co., Ltd；Ethidium bromide (Ethidium bromide, EB) is purchased from
Guangzhou Kang Long bio tech ltd；Agarose is purchased from Invitrogen companies；DMEM, hyclone (Fetal bovine
Serum, FBS), 0.25% trypsase be purchased from GIBCO companies；BIX-01294 is purchased from SIGMA companies；Various cell culture are used
Culture dish, culture plate be purchased from Corning companies；15mL and 50mL centrifuge tubes are purchased from Excell companies；All size
DNase＆RNase-free band filter core suction nozzles and PCR pipe are purchased from Axygen companies；Embryo's injection stage water is purchased from Sigma companies；Group
Antibody used in albumen H3K9me2 and H3K9me3 immunofluorescence is purchased from abcam biotech firms；MTT is purchased from Thermo companies；DAPI
Purchased from Cell Signaling scientific ＆ technical corporation；Q-PCR primers are synthesized by Jin Wei intelligence bio tech ltd.
3rd, main solution is configured
3.1 cells frozen storing liquid：60%DMEM+30%FBS+10%DMSO.
3.2 oocyte maturation culture formula of liquid (M199)：
TCM199+10%pFF+10%FBS+10IU/mL hCG+10IU/mL eCG+0.1mg/mL Cys+
10ng/mL EGF, it is now with the current.
3.3 micromanipulation liquid (HM) are formulated：
0.7696g NaCl,0.0168g NaHCO3,0.0356g KC1,0.0162g KH2PO4,0.0293g MgSO4·
7H2O, 0.1g glucose, 0.0146g glutamine, 0.1501g taurines, 0.2383g HEPES, 0.4g BSA, 0.0065g
Penicillin, 0.0050g streptomysins, Sigma H2O dissolvings are settled to 100mL.Mentioned component is sequentially added into mixing.Adjust pH extremely
7.2~7.4, osmotic pressure is 270~290mOsm, and 0.22 μm of filter filtering is dispensed, and 4 DEG C save backup.
3.4Hoechst33342 formula：10mL pure water dissolves 100mg, and concentration is 10mg/mL, and -20 DEG C freeze.
4th, key instrument equipment
Trace dna concentration meter (Nanodrop 2000, Thermo, the U.S.)
Quantitative PCR apparatus (ECOTM, Illumina, the U.S.)
Micropipettor (Eppendorf, Germany)
Electric-heated thermostatic water bath (DK-S24, Shanghai is gloomy reliable to test Instrument Ltd.)
PCR instrument (Hangzhou BIOER Technology Co., Ltd, China)
Horizontal electrophoresis tank and electrophoresis apparatus (Beijing Liuyi Instrument Factory, China)
Gel imaging system (UVP, the U.S.)
- 80 DEG C of ultra low temperature freezers (Thermo, the U.S.)
Superclean bench (SuZhou Antai Air Tech Co., Ltd., China)
The ultrapure water purification systems of MILLI-Q (Millipore companies, the U.S.)
Centrifuge 5810R refrigerated centrifuges (Eppemdorf, Germany)
Forma 3111,3131 CO2gas incubators (Thermo, the U.S.)
TE300 and TE2000U inverted microscopes (Nikon, Japan)
Micromanipulation instrument (Narishige, Japan)
The supporting thermal station of stereomicroscope (Tokai, Japan)
Electric heating constant-temperature blowing drying box：It is gloomy reliable to test Instrument Ltd., Shanghai
Stereoscopic thermal station：East Sea company (Tokai), Japan
Draw pin instrument, PN-30, Narishige, Japan
Micro- disconnected instrument, MF900, Narishige, Japan
Beveller, EG-400, Narishige, Japan
Magnetic stirring apparatus：Haimen City its woods Bel's instrument manufacturing Co., Ltd, Shanghai
Electronic analytical balance：AC11.4, Sai Duolisi scientific instrument (Beijing) Co., Ltd, China
5th, analysis software and website
PCR primer is designed and analysis software：primer premier5.0,genetool
DNA sequence data storehouse：NCBI/GenBank/nucleotide/UCSC/Ensembl
Picture processing：Photoshop 7.0/ACDSee 9.0
Gel imaging system software：BioCaptMw
Genetic analysis software：MEGA5.05
Experimental method
1st, reverse transcription and quantitative PCR
Cell RNA progress reverse transcription is obtained into cDNA, specific experiment method is with reference to upset record kit TAKARA
Reverse Transcription Kit specifications are carried out.Thaw cell RNA on ice, while under the conditions of room temperature (15-25 DEG C)
Defrosting Reverse Transcription；Genomic DNA is removed, by the composition of table 1 in preparing reaction mixture on ice, in order to ensure reaction mixture
The adequacy of preparation, should be carried out Master Mix preparation by the amount of stoichiometric number+2 when preparing, be incubated under the conditions of 42 DEG C
2min。
Table 1 removes genomic DNA reaction system
Reverse transcription reaction is carried out again.Reaction system is prepared according to table 2, all operations are carried out on ice.
Table 2RNA reverse transcription reaction systems
Response procedures：7 DEG C, 15min；85 DEG C, 5s.Reacted in PCR instrument.React reverse transcription product 4 after terminating
It is DEG C of short duration to preserve, it is -20 DEG C long-term to preserve or carry out quantitative RT-PCR detection.
The real-time quantitative PCR reaction system of table 3
Real-time quantitative PCR uses quantitative reaction kit (the SYBR Select of Life Technologies companies
Master Mix) to be tested, reaction system is 10uL (see above-mentioned table 3), and each 3-4 technology is repeated.PCR response procedures
For：95 DEG C of pre-degenerations, thermal starting 5min；45~50 wheel PCR cycles (95 DEG C, 10s, 60 DEG C, 15s, 72 DEG C, 20s)；Solubility curve
(95 DEG C, 15s, 55 DEG C, 15s, 95 DEG C, 15s).Quantitative primer is shown in Table 4.
The quantification PCR primer of table 4 is designed
2nd, data processing and statistical analysis
In gene relative expression data analysis, each gene C t values are by reference gene (β-actin) and control sample normal state
After change processing, with 2-△△CtMethod calculates relative expression quantity.Specific practice is that every group of sample target gene Ct average is subtracted into internal reference
Gene C t is worth to △ Ct values, and using one of which sample △ Ct values as reference, other processing sample △ Ct values are subtracted into ginseng
The △ △ Ct values of each group sample are worth to according to group sample △ Ct, finally the purpose base by each processing sample with respect to reference group sample
Because expression quantity uses 2-△△CtRepresent.Data processing is carried out using SPSS17.0, significance test is carried out using Chi-square Test.Probability
Value P<When 0.05, average value significant difference.
First, clone embryos treatment fluid is to pig adult fibroblast cloning and its early embryonic development ability comparative experiments
The preparation of PZM nutrient solutions (embryo medium)：
Take 200mL Sigma H2O, is sequentially added into following component：3.156g NaCl,1.053g NaHCO3,0.373g
KC1,0.024g KH2PO4,0.049g MgSO47H2O, 0.308g calcium lactate, 0.011g Sodium Pyruvates, 0.073g L- paddy
Glutamine, 0.273g hypotaurine, 10mL BME, 5mL MEM, 0.033g penicillin, 0.025g streptomysins adjust pH after mixing
For 7.2~7.4, osmotic pressure is 288 ± 2mOsm, is settled to 500mL, 4 DEG C save backup.
The BIX-01294 hydrochlorides for weighing amount of calculation are added to after the dissolving of PZM nutrient solutions, you can obtain clone embryos processing
Liquid.
1st, the collection of egg mother cell and In-vitro maturation
From collection pig ovary is butchered, it is put into the vacuum flask containing 1% 28-39 DEG C of dual anti-physiological saline, is sent back in 4h
Laboratory.Cleaned up first with 39 DEG C of physiological saline, then extract a diameter of 4- with the 10ml syringes of No. 18 syringe needles of band
Egg mother cell in 6mm ovarian follicles, extract stands 15min in 39 DEG C of water-baths, goes after supernatant to add DPBS resuspensions, stands
Supernatant is removed after 2-3min and is repeated once.Pick out that cytoplasm is uniform, ovarian cumulus is fine and close under Stereo microscope and 2 layers of parcel with
On cumulus cell-oocyte complex (Cumulus oocyte complexes, COCs), with DPBS and M199 maturation train
Nutrient solution is washed after 3 times respectively, is transferred to the M199 maturation trainings that 2h has been balanced in incubator and embryo's level mineral oil is covered
In nutrient solution drop or in four orifice plates.39 DEG C, maturation culture 42h in the incubator of 5%CO2 saturated humidities.
2nd, granular cell is removed
The COCs of maturation culture is transferred in the centrifuge tube containing hyaluronidase, is transferred to after pipettor piping and druming about 2min
In 30mm culture dishes, the egg mother cell for sloughing ovarian cumulus is sorted out with mouth suction pipe.Egg mother cell is washed, chooses under body formula mirror and carries
The mature oocyte of first polar body is standby.
3rd, the pretreatment of donorcells
In order to improve the fusion rate of reconstructed embryo, serum starvation and contact inhibition processing need to be done to donorcells.Recovery cell
Afterwards, when cell confluency degree reaches more than 95%, the DMEM medium cultures without serum is used instead and are stayed overnight.Secondary daily digesting, uses HN
Operate liquid that the cell after centrifugation is resuspended into piping and druming uniformly, prepare clone.
4th, pig adult fibroblast cloning
Select and discharged first polar body and the good egg mother cell of form, with 100-120 μm of fixed pin of external diameter, internal diameter is
15-20 μm of kernel removing needle is enucleated using blind suction method.Comprise the following steps that：The μ L sizes of 4 drop 50 are done in 65mm sterile culture disks
Operate in drop, paraffin oil covering moves into the egg mother cell of 30 or so and appropriate body cell wherein, with fixed pin gently
Egg mother cell is fixed, egg mother cell is stirred with kernel removing needle makes polar body in the position at general 5 o'clock.Then with kernel removing needle along 3 points
Position is lunged, and removes polar body and neighbouring kytoplasm, then pin is withdrawn from, and polar body and kytoplasm are spued, and is subsequently injected into one big
Small suitable, the smooth body cell in surface completes embryo's restructuring procedure.Reconstructed embryo is put into renewal cultivation 1h in embryo medium.
Renewal cultivation 1h reconstructed embryo is transferred in fusion liquid after balance 2min in batches, 3 are washed with fusion/activation liquid
Time, then 5~8 every batch be put into be paved with fusion liquid integration slot in, stir reconstruct with drawing, superfine solid glass pin
Ovum, makes donor cell and the cell membrane contact face of receptor oocytes and electrode runs parallel, applies 150v/mm, 100 μ s', 2DC is straight
The electric pulse induced fusion of stream, activation reconstruct embryo, then wash the embryo for being transferred to mineral oil covering after 3 times immediately with embryo medium
In tire nutrient solution, 39 DEG C, 5%CO are placed in2, saturated humidity incubator in cultivate, after 4h under stereomicroscope decision fusion
Situation simultaneously calculates fusion rate.
5th, using the embryo 23-24h after clone embryos treatment fluid culture 24h
BIX-01294 hydrochlorides are added in PZM nutrient solutions to be configured at 5nM, 50nM, 500nM clone embryos respectively
Manage liquid.The reconstructed embryo 5 times of fusion is washed with PZM nutrient solutions, is transferred in the good nutrient solution of pre-equilibration, be placed in 39 DEG C, saturated humidity,
Hypoxemia (5%O2+ 5%CO2+ 90%N2) condition incubator in cultivate.It is designated as when embryo fusion is activated 0 day, by clone embryos
Treatment fluid was transferred in good new four orifice plate of pre-equilibration respectively the 1st day 8 points of morning, and clone embryos treatment fluid processing time is 23-
24h, the 2nd day 8 points of morning terminates the drug exposure times, and embryo change and continues to cultivate after liquid.Drug-treated it is different dense
Spend packet situation such as following table table 5.Observe respectively within 2nd day and the 6th day and record the cloning efficiency dependency number such as spilting of an egg number and blastaea number
According to.
The clone embryos treatment fluid of table 5 handles embryo's 23-24h groups
6th, clone embryos treatment fluid processing embryo 16-17h
The reconstructed embryo 5 times of fusion is washed with nutrient solution, is transferred in the good nutrient solution of pre-equilibration, be placed in 39 DEG C, saturated humidity,
Cultivated in the incubator of hypoxemia (5%O2+5%CO2+90%N2) condition.0 day, embryo fusion are designated as when embryo fusion is activated
It is transferred to respectively after 4h in good new four orifice plate of pre-equilibration, clone embryos treatment fluid processing time is 16-17h, then terminates the medicine
Thing processing time, embryo change and continues to cultivate after liquid.The various concentrations packet situation such as table 6 below of drug-treated.2nd day and
The cloning efficiency related data such as spilting of an egg number and blastaea number is observed and recorded respectively within 6th day.
The clone embryos treatment fluid of table 6 handles embryo's 16-17h groups
Two schemes were cultivated to the 6th day morning, 5-10 blastaea were collected every time for every group and carried out in nuclear staining statistics blastaea
Total cell number, after HN operation liquid washings, is put into lucifuge in the DPBS containing 10 μ g/mL Hoechst33342 and dyes 10min, connect
And blastaea droplet is transferred on clean slide, light covered makes blastaea be expanded after being pressurized but be unlikely to broken, used
Nail sheet for oil seal.Fluorescence microscopy Microscopic observation is placed in, the total cell number in single blastaea is calculated.In addition, every group every time in addition
Collect 6-10 embryo's mixing to be filled in 50 μ L RLT lysates, cover centrifugation lid, sealed, lost rapidly with Parafilm films
Enter quick-frozen in liquid nitrogen, -80 DEG C of preservations, dry ice transport, for embryo RNA extractings.
Data processing is carried out using SPSS17.0, compares control group and 5nM, 50nM, 500nM BIX- using Chi-square Test
01294 treatment group reconstructs the initial in vitro developmental state of embryo, including fusion rate, cleavage rates, blastocyst rate and capsule after being cloned
The difference of embryo average cell number.Probable value P<When 0.05, mean difference is notable.
2nd, embryo's specific gene expression is detected after the processing of clone embryos treatment fluid
1st, the collection of embryo
Be ready to equipped with 50 μ L RLT lysates centrifuge tube, development to during blastaea from four orifice plates take out 6-10 embryo
Tire is put in centrifuge tube, is sealed with Parafilm films, is lost quick-frozen in liquid nitrogen rapidly, -80 DEG C of preservations.
2nd, embryo RNA is extracted
The embryo of collection extracts for RNA, and embryo RNA extraction uses Qiagen RNeasy Plus Micro Kit
(50) kit.Concrete operations are as follows：
(1) embryo samples are pre-processed.39 DEG C of water-baths 1~2min of defrosting, simple centrifugal.5 μ L, 4ng/ are added into lysate
μ L carrier RNA, add 1 μ L beta -mercaptoethanols, mix, vortex 30s, simple centrifugal.
(2) premixed liquid is shifted to AllPrep DNA spin column, 8000 × g or >=10,000rpm centrifugations 30s.
(3) 70% isometric ethanol solution is added in collecting pipe in filtrate in (2), piping and druming is mixed.
(4) alcohol mixeding liquid of collecting pipe in (3) is transferred to RNeasy Min Elute spin column, 8000
× g or >=10,000rpm centrifugation 15s, outwell filtrate.
(5) 15s is centrifuged to adding 700 μ L Buffer RW1,8000 × g or >=10,000rpm in pillar, outwell filtrate.
(6) 15s is centrifuged to adding 500 μ L Buffer RPE, 8000 × g or >=10,000rpm in pillar, outwell filtrate.
(7) to the ethanol of 500 μ L 80% is added in pillar, 15s is centrifuged, pillar is carefully taken out, discards collecting pipe.
(8) pillar is installed to a new 2mL collecting pipe, is spaced apart, opens lid, at full speed centrifugation 5min.
(9) eluted rna：Pillar is installed to the 1.5mL collecting pipes that kit is carried, by 14 μ L RNase-free water
The center of centrifugal column film is fed directly to, froth lid centrifuges 1min at full speed, and -80 DEG C preserve RNA or carry out reverse transcription at once.
Embryo RNA reverse transcriptions and quantitative PCR are carried out according to step 2.3.2.6, detection embryo specific gene (DNMT1,
DNMT3a, DNMT3b, NANOG, SOX2, CDX2, BMP4, ESRRB, KDM1a, XIST, OCT4) expression.Compare each group
Clone the difference of embryo specific gene expression quantity.Quantitative primer is shown in Table 7.
The embryo's quantification PCR primer of table 7 is designed
3rd, data processing and statistical analysis
In genome relative expression data analysis, each gene C t values are by reference gene (β-actin) and control sample is just
After stateization processing, with 2-ΔΔCtMethod calculates relative expression quantity.Data processing is carried out using SPSS17.0, Chi-square Test is utilized.Generally
Rate value P<When 0.05, mean difference is notable.
3rd, histone H 3 K9me2 expressions in immuno-fluorescence assay clone embryos
The blastaea of collection in 6th day is put into effect 1min zona-frees in 0.5% pronase of preheating, removed saturating
3 times, 15min are washed after oolemma with DPBS-0.1%PVA；Add fixative (4%PFA diluted with DPBS), incubation at room temperature
3 times, 15min are washed after 15min with DPBS-0.1%PVA；Add perforation liquid (0.5%TritionX-100 diluted with DPBS)
3 times, 15min are washed with DPBS-0.1%PVA after incubation at room temperature 30min；Add confining liquid (1%BSA diluted with DPBS), 4
DEG C overnight.Washed after adding the primary antibody dilution diluted with 1%BSA, 39 DEG C of incubation 1h with DPBS-0.1%PVA 12 times,
60min, adds after 39 DEG C of secondary antibody dilution (noting lucifuge processing) is incubated 1h and is washed with DPBS-0.1%PVA 12 times,
60min.It is eventually adding after 1 μ g/mL PI, incubation at room temperature 10min with DPBS-0.1%PVA several times.Get out clean load glass
Piece, the embryo transfer dyed after treating is got on, and makes operation quick in transfer process as far as possible and carrying liqs less of trying one's best, with embryo
Centered at where tire, corner is found around the embryo of cover glass to be placed, vaseline/paraffin oil is dripped, covered,
Light cap slide, then uses nail sheet for oil seal, finally in Nikon E800 fluorescence microscopy Microscopic observations, photographs to record.
Analysis of experimental results
1st, clone embryos treatment fluid handles the influence to pig adult fibroblast neural specific gene expression
Using the clone embryos treatment fluids of various concentrations, (BIX-01294 is dense after the 5th generation pig adult fibroblast is adherent
Degree be respectively 0,5nM, 50nM, 500nM) respectively processing 24h after collect cell, with OMEGA Total RNA Kit I kits
Extracting RNA is simultaneously inverted to cDNA；Pig adult fibroblast cellular histone methylase related gene is detected with q-PCR methods
The mRNA expressions of (SUV39H1, G9a, SETdb1).
As a result such as Fig. 1 is shown, different explanation significant difference a, b, the c (P of letter in Fig. 1<0.05).As illustrated, using
BIX-01294 concentration is that 5, the SUV39H1 gene expressions of 50nM clone embryos treatment fluid treatment group cell are substantially less than nowhere
Manage control group (P<0.05), and 500nM treatment groups are then significantly higher than remaining each group (P<0.05)；5th, at 50nM BIX-01294
Manage after cell, G9a expression quantity has elevated trend, and 500nM treatment group G9a gene expressions are significantly higher than without processing control
Group (P<0.05)；After being handled through 0,5nM, 50nM, 500nM BIX-01294, SETdb1 expression quantity is substantially less than without processing
Control group (P<0.05).
2nd, DNA methylation enzyme related gene
DNA methylation level is main to be acted on by the mutual Coordinated Play of DNA methylation transferase.Therefore, for clone
Whether cell DNA methylates and reprograms embryo's treatment fluid before and after the processing, in the cells of BIX-01294 before and after the processing
DNMT1, DNMT3a, DNMT3b mRNA level in-site have carried out q-PCR and have detected and be compared.In the 5th generation pig adult fibroblast
Located respectively using the clone embryos treatment fluid (BIX-01294 concentration is respectively 0,5nM, 50nM, 500nM) of various concentrations after adherent
Cell is collected after reason 24h, kit extracting RNA is simultaneously inverted to cDNA；Pig adult fibroblast DNA first is detected with q-PCR methods
Base enzyme related gene DNMT1, DNMT3a, DNMT3b mRNA expressions.
As a result as shown in Fig. 2 through 5nM, 50nM, 500nM BIX-01294 clone embryos treatment fluid processing after, DNMT1
Expression quantity be substantially less than untreated control group (P<, and the DNMT1 expression quantity of 5nM, 50nM treatment group is significantly reduced 0.05)
(P<0.05)；After being handled through 5nM, 50nM, 500nM BIX-01294, DNMT3a expression quantity is substantially less than untreated control
Group (P<, and the DNMT1 expression quantity pole of 50nM treatment groups significantly reduces (P 0.05)<0.01)；5nM BIX-01294 handle cell
DNMT3b gene expression doses are made to be substantially less than untreated fish group (P<0.05), the DNMT3b gene tables of 50nM, 500nM treatment group
Up to the trend for having reduction compared with untreated fish group.
3rd, influence of the clone embryos treatment fluid to histone H 3 K9 di-methylations
In order to study the histone methylated modification of porcine somatic cell cloning embryos embryo in somatic cell clone clone embryos early development mistake
Influence in journey, therefore renewal cultivation after clone embryos 24h is handled with the clone embryos treatment fluid of various concentrations, in embryonic development
H3K9me2 expressions are detected to embryo is collected during 2cell and 4cell.
Immunofluorescence results are as shown in figure 3, clone embryos treatment fluid is to clone embryos 2cell, 4cell H3K9me2 water
It is flat that there is obvious downward effect.
4th, the comparison of clone embryos neural specific gene expression amount
4.1 histone methylase genes
In order to which whether the processing of clone embryo treatment fluid changes the expression of embryo's histone methylase related gene,
Clone embryos 23-24h after 5nM, 50nM, 500nM BIX-01294 clone embryos treatment fluid processing culture 24h, and in body
Outer culture is developed to blastocyst stage and collects embryo 5-6 in RTL lysates, with Qiagen Qiagen RNeasy Plus
Micro Kit (50) kit extracting RNA is simultaneously inverted to cDNA；The blastaea histone methyl collected for the 6th day is detected with q-PCR methods
Change the mRNA expressions of enzyme related gene (SUV39H1, G9a, SETdb1).
As a result as Fig. 4 shows that the SUV39H1 gene expressions of the clone embryos treatment fluid treatment group embryo of various concentrations have
Declined, and the SUV39H1 gene expressions of 50nM, 500nM treatment group embryo are substantially less than without processing control group (P<0.05)；
The G9a gene expressions of 5nM, 50nM BIX-01294 treatment group embryos have a declining tendency, and 500nM treatment group G9a genes
Expression has elevated trend compared with untreated control group；After being handled through 5nM BIX-01294, SETdb1 expression quantity is significantly higher than not
Handle control group (P<0.05), and 50, the SETdb1 gene expressions of 500nM BIX-01294 treatment group embryos have becoming for decline
Gesture.
4.2DNA methylases gene
Mrna expression amount result is as shown in figure 5, at the clone embryos treatment fluid through 5nM, 50nM, 500nM BIX-01294
After reason, DNMT1 expression quantity pole is significantly higher than untreated control group (P<0.01), and with the increase of concentration for the treatment of, DNMT1 bases
Because of expression quantity increase；After being handled through 5nM, 50nM, 500nM BIX-01294, DNMT3a expression quantity pole is significantly higher than untreated
Control group (P<0.01), and with the increase of concentration for the treatment of, the increase of DNMT3a gene expression amounts；5th, 50nM BIX-01294 processing
Cell makes DNMT3b gene expression doses be had a declining tendency compared with untreated control group, and the DNMT3b genes of 500nM treatment groups
Express the trend being improved compared with untreated control group.
Above-described is only some embodiments of the present invention.For the person of ordinary skill of the art, not
On the premise of departing from the invention design, various modifications and improvements can be made, these belong to the protection model of the present invention
Enclose.
Claims (8)
1. a kind of clone embryos treatment fluid, it is characterised in that be made up of BIX-01294 and PZM nutrient solutions, the PZM nutrient solutions
Described in BIX-01294 concentration be 5nM~500nM；Wherein, the PZM nutrient solutions are composed of the following components：
2. clone embryos treatment fluid according to claim 1, it is characterised in that the clone embryos treatment fluid pH is 7.2
~7.4, osmotic pressure is 288 ± 2mOsm.
3. clone embryos treatment fluid according to claim 1, it is characterised in that the concentration of the BIX-01294 is 50nM
~500nM.
4. a kind of application method of clone embryos treatment fluid according to claim 1, it is characterised in that including following step
Suddenly：
(1) pig ovary is collected, is put into the vacuum flask containing 1% 28-39 DEG C of dual anti-physiological saline, first with 39 DEG C of physiology
Salt solution is cleaned up, and the egg mother cell in a diameter of 4-6mm ovarian follicles is then extracted with the 10ml syringes of No. 18 syringe needles of band, is extracted
Liquid stands 15min in 39 DEG C of water-baths, goes after supernatant to add DPBS resuspensions, stands and supernatant and repetition one are gone after 2-3min
It is secondary, finally pick out that cytoplasm is uniform, ovarian cumulus is fine and close under Stereo microscope and cumulus cell-ovum of more than 2 layers of parcel is female carefully
Born of the same parents' complex is washed after 3 times respectively with DPBS and M199 maturation culture solutions, is transferred to and 2h has been balanced in incubator and has been covered
In the M199 maturation cultures drop of embryo's level mineral oil or in four orifice plates, in 39 DEG C, the incubator of 5%CO2 saturated humidities
Maturation culture 42h.
(2) cumulus cell-oocyte complex of maturation culture is transferred in the centrifuge tube containing hyaluronidase, moved
It is transferred in 30mm culture dishes, is sorted out the egg mother cell for sloughing ovarian cumulus after liquid device piping and druming about 2min with mouth suction pipe, washing ovum is female
Cell, the mature oocyte with first polar body is chosen under body formula mirror.
(3) by the mature oocyte, with 100-120 μm of fixed pin of external diameter, internal diameter uses blind for 15-20 μm of kernel removing needle
Suction method is enucleated, and is comprised the following steps that：Drop 4 drips the operation drop of 50 μ L sizes in 65mm sterile culture disks, is covered using paraffin oil
Lid, then the egg mother cell of 30 or so and appropriate body cell are moved into wherein, egg mother cell is gently fixed with fixed pin, is used
Kernel removing needle, which stirs egg mother cell, makes polar body in the position at general 5 o'clock, is then lunged with kernel removing needle along 3 positions, removes depolarization
Body and neighbouring kytoplasm, then withdraw from pin, and polar body and kytoplasm are spued, and are subsequently injected into a size properly, surface is smooth
Body cell, completes embryo's restructuring procedure.Reconstructed embryo is put into renewal cultivation 1h in embryo medium；
(4) reconstructed embryo is transferred in fusion liquid after balance 2min in batches, then 5~8 every batch are put into and have been paved with fusion
In the integration slot of liquid, reconstructed eggs are stirred with solid glass pin, make the cell membrane contact faces of donor cell and receptor oocytes with
Electrode runs parallel, applies 150v/mm, 100 μ s, 2DC electric pulse induced fusion, activation reconstruct embryo are then trained with embryo
Nutrient solution wash 3 times after be transferred to immediately mineral oil covering embryo medium in, be placed in 39 DEG C, 5%CO2, the culture of saturated humidity
Cultivated in case.
(5) wash with nutrient solution the reconstructed embryo 5 times of fusion, be transferred in the good nutrient solution of pre-equilibration, be placed in 39 DEG C, saturated humidity,
Cultivated in the incubator of hypoxia condition, continuation culture is carried out to embryo using the clone embryos treatment fluid.
5. application method according to claim 4, it is characterised in that the hypoxia condition in the step (5) refers to described
Air composition in incubator is 5%O2+ 5%CO2+ 90%N2。
6. application method according to claim 4, it is characterised in that further comprising the steps of before the step (5)：
After recovery cell, when cell confluency degree reaches more than 95%, use the DMEM medium cultures without serum instead and stay overnight.It is secondary
Daily digesting, operates liquid that the cell after centrifugation is resuspended into piping and druming uniformly, prepares clone with HN.
7. clone embryos treatment fluid according to any one of claims 1 to 3 reduction cell SUV39H1, SETdb1,
Application in terms of DNMT1 or DNMT3a gene expression.
8. the clone embryos treatment fluid according to any one of claims 1 to 3 is lowering the early embryo development stage
The application of 2cell, 4cell H3K9me2 levels.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710339889.XA CN107227298B (en) | 2017-05-15 | 2017-05-15 | Cloned embryo treating fluid, use method thereof and application of treating fluid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710339889.XA CN107227298B (en) | 2017-05-15 | 2017-05-15 | Cloned embryo treating fluid, use method thereof and application of treating fluid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107227298A true CN107227298A (en) | 2017-10-03 |
| CN107227298B CN107227298B (en) | 2021-06-25 |
Family
ID=59933216
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710339889.XA Active CN107227298B (en) | 2017-05-15 | 2017-05-15 | Cloned embryo treating fluid, use method thereof and application of treating fluid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107227298B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234225A (en) * | 2018-11-02 | 2019-01-18 | 温氏食品集团股份有限公司 | A kind of embryo medium and a kind of processing method of somatic cell clone embryo |
| CN109406471A (en) * | 2018-10-26 | 2019-03-01 | 安徽农业大学 | A kind of fluorescence imaging method with embryo's stereochemical structure that vaseline column is support |
| CN109536610A (en) * | 2018-12-05 | 2019-03-29 | 西南大学 | Application of the SETDB1 in regulation cholesterol approach |
| CN110373416A (en) * | 2019-06-18 | 2019-10-25 | 华南农业大学 | Application of the RBP1 gene in sow gonad granulocyte |
| CN111760034A (en) * | 2020-06-29 | 2020-10-13 | 华南农业大学 | Application of DNMT1 gene in porcine ovarian granulosa cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002157A (en) * | 2015-07-06 | 2015-10-28 | 广东温氏食品集团股份有限公司 | Porcine somatic cell nucleus transplanting fusion method |
| CN106520838A (en) * | 2016-10-24 | 2017-03-22 | 湖北省农业科学院畜牧兽医研究所 | New method for gene injection for somatic cell nuclear transfer reconstructed embryo |
-
2017
- 2017-05-15 CN CN201710339889.XA patent/CN107227298B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002157A (en) * | 2015-07-06 | 2015-10-28 | 广东温氏食品集团股份有限公司 | Porcine somatic cell nucleus transplanting fusion method |
| CN106520838A (en) * | 2016-10-24 | 2017-03-22 | 湖北省农业科学院畜牧兽医研究所 | New method for gene injection for somatic cell nuclear transfer reconstructed embryo |
Non-Patent Citations (2)
| Title |
|---|
| JIAOJIAO HUANG等: "BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei", 《REPRODUCTION》 * |
| 李果: "抑制组蛋白H3K9me3甲基化对猪克隆胚胎体外发育效率的影响", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406471A (en) * | 2018-10-26 | 2019-03-01 | 安徽农业大学 | A kind of fluorescence imaging method with embryo's stereochemical structure that vaseline column is support |
| CN109234225A (en) * | 2018-11-02 | 2019-01-18 | 温氏食品集团股份有限公司 | A kind of embryo medium and a kind of processing method of somatic cell clone embryo |
| CN109234225B (en) * | 2018-11-02 | 2022-03-11 | 温氏食品集团股份有限公司 | Embryo culture solution and method for processing somatic cell cloned embryos |
| CN109536610A (en) * | 2018-12-05 | 2019-03-29 | 西南大学 | Application of the SETDB1 in regulation cholesterol approach |
| CN110373416A (en) * | 2019-06-18 | 2019-10-25 | 华南农业大学 | Application of the RBP1 gene in sow gonad granulocyte |
| CN110373416B (en) * | 2019-06-18 | 2022-09-20 | 华南农业大学 | Application of RBP1 gene in sow ovarian granulosa cells |
| CN111760034A (en) * | 2020-06-29 | 2020-10-13 | 华南农业大学 | Application of DNMT1 gene in porcine ovarian granulosa cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107227298B (en) | 2021-06-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107227298A (en) | A kind of clone embryos treatment fluid and its application method and the purposes of the treatment fluid | |
| KR102671098B1 (en) | Cellular microcompartment and preparation methods | |
| CN104312971A (en) | Method for promoting in-vitro buffalo oocyte maturation | |
| CN105524940A (en) | Vector, cell and method for improving bovine cloning efficiency on the basis of histone methylation modifying level | |
| CN108504625A (en) | A kind of l cell and application thereof | |
| CN103952368B (en) | A kind of culture fluid promoting porcine somatic cell cloning embryos vitro Development of Embryos | |
| CN103993027B (en) | A kind of method that transgene pig riddled basins are knocked out | |
| Al-Ghadi et al. | The in vitro development of cloned sheep embryos treated with Scriptaid and Trichostatin (A) | |
| CN103409367A (en) | Method for improving quality of external fertilization embryo of oocyte of sheep | |
| CN104513807B (en) | The method for cloning non-human animal is separated, cultivates the method for cell and carried out from blood | |
| CN101974565B (en) | Method for producing transgenic buffalo embryos by applying intracytoplasmic sperm injection (ICSI) mediation | |
| CN106591374B (en) | A method of improving pig nucleus transplantation embryonic development efficiency | |
| CN105349485B (en) | Improved method for cutting buffalo blastocyst by bare hand and cutting fluid | |
| CN103725644B (en) | Cherry valley duck embryo epithelial cell line and method for building up thereof | |
| CN109234225B (en) | Embryo culture solution and method for processing somatic cell cloned embryos | |
| KR20080077738A (en) | A methods to improve in vitro maturation of pig oocytes | |
| Picton | Therapeutic Potential of In Vitro–Derived Oocytes for the Restoration and Treatment of Female Fertility | |
| KR102004958B1 (en) | Method for isolating and culturing alive cells from fossil or curcass of ancient extinct creature | |
| CN111286485B (en) | Application of caprylated Ghrelin in inhibiting secretion of cumulus cell complex cAMP and MAPK in vitro | |
| CN112226404B (en) | Culture medium composition and culture method for promoting animal embryo in-vitro development | |
| Rakotondrafara et al. | Preparation and electroporation of oat protoplasts from cell suspension culture | |
| CN103409467B (en) | Method for acquiring heterogeneity interspecies-cloned yak premium embryos | |
| CN103509752B (en) | Hubei white pig fetal fibroblast cell line | |
| CN108118027A (en) | Sheep oocyte in-vitro 'two-stage' maturation method, and pre-incubation liquid and kit thereof | |
| CN113249304B (en) | Application of sulforaphane in preparation of bovine oocyte in-vitro maturation liquid |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |